1,181 Matching Annotations
  1. May 2019
    1. debris, polysaccharides, and high molecular weight DNA The supernatant was gently decanted into a fresh microcentrifuge tube and 200!!L of chloroform/ mL of TRizol was added and the tube was shaken vigorously for 15s. The mixture was incubated at room temperature for 2-3 min before centrifugation at 12000 x g for 15 min at 4 °C. This resulted in the separation of the mixture into a lower organic phase and an upper aqueous phase. The aqueous phase containing the RNA was gently aspirated and transferred into a fresh microcentrifuge tube and 500!!L of isopropanol/ mL of TRizol reagent was added and incubated at RT for 10min. The mixture was centrifuged at 12000 x g for 10 min at 4 °C to isolate the RNA as a pellet. The supernatant was discarded and the pellet was washed once with 70% ethanol, centrifuged and the pellet was air-dried and re-dissolved in approximate quantity of nuclease free (DEPC-treated) water. The purity (A2so/ A260 >1.8) and concentration (A2soX dilution factor X 40) of the obtained RNA was determined by measuring the absorbance at 260nm (A26o) and 280nm (A2so). For storage, the RNA was resuspended in 1mL of absolute ethanol and stored at -70°C. Subsequently before use, the RNA was pelleted at 12000 x g for 10 min at 4°C, washed with 70% ethanol and redissolved in DEPC-treated water.
    2. Total RNA was isolated from cells using TRizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. Briefly 2X108 cells were harvested by centrifugation at 1258 x g for 10 min, and washed 1X with PBS. The cell pellet was lysed with 2 mL ice-cold TRizol reagent. The lysate was centrifuged at 12000 x g for 10 min at 4 °C to pellet down cellular
    3. Total RNA isolation
    4. Molecular Biology Techniques
    5. x g for 10 min at RT. The supernatant was centrifuged at 1258 x g for 10 min at RT. The pellet obtained was washed 2X at 4°C in half the culture volume of Cytomix buffer (120 mM KCl, 0.15 mM CaCh, 10 mM K2HP04, 25 mM HEPES, 2 mM EDTA, and 2mM MgCh; pH 7.6) and then resuspended in chilled cytomix buffer at a density of 2 X 108 cells/mL. Electroporation: For a single electroporation, 2011g of plasmid (in water or 10mM Tris pH 8.0) for episomal expression and 5Jlg of plasmid for integration events, was added to a pre-chilled cuvette ( 4mm, BTX, San Diego, CA). 500J1L of chilled cell suspension (108 cells) processed as above was transferred to the cuvette and mixed with DNA by gently tapping and incubated on ice for 10 min. The cells were electroporated twice at 25 J.IF, 1500 V (3.75 kV /em), pausing 10 s between pulses (Robinson and Beverley, 2003) in a BioRad Gene Pulser X Cell electroporator. The cell suspension was then transferred to 5mL of mDMEM containing 20% FBS and allowed to recover for ~18hrs before antibiotic selection commenced. Selection of transformants: Selection of parasites containing recombinant DNA was carried out initially in liquid medium followed by culture with agar. After the rest period, the electroporated cells were exposed to 1011g/ mL of G418 or Hygromycin B for 48 hrs with antibiotic being increased to 2011g/ mL and 50Jlg/ mL at 48 hr. intervals. Part of the cells were then plated onto freshly poured mDMEM plates (1X mDMEM, 2% agar containing 100]lg/ mL, 500]lg/ mL or 1mg/ mL G418; or 50]lg/ mL Hygromycin B) and incubated at 23°C for 7-14 days. Individual colonies obtained on plates were cultured in liquid medium and screened appropriately. Limiting dilution was used to isolate single clones.
    6. Preparation of Leishmania culture for electroporation: An early log phase culture of Leishmania donovani was harvested and dead cells pelleted at 129
    7. Foreign DNA can be introduced into Leishmania cultures using electroporation. Transfected circular plasmids are maintained as episomes, while linear DNA can integrate into the genome. Preparation of DNA : DNA construct to be electroporated was generated using standard molecular biological techniques as described later. The plasmid DNA was prepared from E. coli DH5-a or XL-1 Blue cells using the EndoFree MaxiPrep kit from Qiagen (Hilden, Germany) according to manufacturer's protocol. Briefly, 200mL of overnight culture was pelleted at 6000 x gat 4°C for 15 min. The pellet was resuspended in 10mL Buffer P1 (50mM Tris-Cl, pH 8.0, 10mM EDTA, 100p.g/mL RNaseA). To this 10mL Buffer P2 (200mM NaOH, 1 %w /v SDS) was added and mixed thoroughly by vigorously inverting 4-6 times and an incubated at RT for 5 mins. Now 10mL of chilled Buffer 3 (3.0M Potassium acetate, pH 5.5) was added and mixed thoroughly by vigorously inverting 4-6 times and then the lysate was poured into a QIAfilter catridge and incubated at RT for 10 min. Subsequently a plunger was used to filter the cell lysate into a 50mL tube to which 2.5mL Buffer ER was added and inverted 10 times to mix. This was incubated on ice for 30 min. In the meantime, a QIAGEN-tip was equilibrated with 10mL Buffer QBT (750mM NaCl, 50mM MOPS, pH 7.0, 15%v /v isopropanol and 0.15%v jv Triton X-100). After incubation, the filtrate was allowed to enter the tip resin by gravity. This was followed by two washes with 30mL of Buffer QC (1.0M Nacl, 50mM Tris-Cl, and pH 7.0 and 15% v /v Isopropanol). DNA was eluted with 15mL Buffer QN (1.6M NaCl, 50mM MOPS, pH 7.0 and 15%v /v isopropanol) and precipitated by adding 10.5 mL (0.7 volumes) isopropanol at RT and centrifugation at 15,000 x g for 30 min at 4°C. The pellet was washed with 5mL endotoxin-free 70% ethanol at RT and centrifuged at 15000 x g for 10 min. The pellet obtained was air dried for 5-10 min and redissolved in a suitable volume of endotoxin-free buffer TE (10mM Tris-Cl, pH 8.0, 1mM EDTA). The concentration of the obtained DNA was estimated by measuring the absorbance at 260nm (A260) and using the known formula: DNA concentration = A260 X SOX dilution factor.
    8. Transfection of Leishmania donovani promastigotes
    9. Syrian hamsters (Mesocricetus auratus), 3-6 weeks old, were used as in vivo Leishmania infection models. The clinicopathological features of the hamster model of VL closely mimic active human disease. Promastigotes in the stationary phase were harvested at 1258 x g for 10 min at RT and washed several times with sterile PBS to remove all traces of medium and FBS. They were resuspended to a cell count of 2X109 cells/mL in PBS. 10011L of this (108 parasites) were injected intra-cardially into hamsters and infection allowed to proceed for 2 months. After 2 months, the hamsters were euthanized with C02 and the spleens were harvested. The spleens were first weighed, and then cut transversely. The exposed surface was gently pressed onto a clean slide to make imprints which were allowed to air dry. Following this, the smears were fixed either in chilled methanol for 5 min or 4% formaldehyde for 10 min. The slides were washed with PBS and either stored in -70°C for later use or stained with Giemsa stain to visualize infection
    10. For in vitro infection studies; J774A.1 macrophages were plated on coverslips at the density of 1 X105 cells/ coverslip in a 6-well culture plate or at a density of 5 X 105 cells/well of a 6-well culture plate, and allowed to rest for 12-18 hrs. The cells were challenged with stationary phase parasites at a multiplicity of infection (MOl) of 1:10 for 6 hrs after which the excess unbound parasites were washed with phosphate buffered saline or plain medium and the macrophages incubated at 37°C for different time points. Infection was visualised by staining the cells with cell permeant nucleic acid stain Syto 11 (Molecular Probes, Eugene, OR) in the dark for 10 min and viewed under a Nikon Eclipse TE2000E fluorescence microscope (Nikon, Japan)
    11. In vivo infection of Syrian hamsters with Leishmania donovani parasites
    12. In vitro infection of J774A.l murine macrophages with L. donovani parasites
    13. 0% FBS and cultured as described above. The cells usually regain motility by 24 to 48 hrs and the culture is ready for use after splitting them once
    14. For long term storage, Leishmania donovani were stored as Dimethyl Sulfoxide (DMSO) containing frozen stocks (freeze downs). Parasites in mid to late log phase were taken and dead and agglutinated cells removed by centrifugation at 129 x g for 10 min. The supernatant was centrifuged at 1258 x g for 10 min to pellet cells which were then washed 2X with plain medium followed by resuspension in FBS containing 10% DMSO as the cryoprotectant. These were transferred to a labelled cryogenic vial and immediately transferred to -20°C for a few hours, followed by incubation at -70°C overnight. The following day, the stocks were transferred to liquid nitrogen (liq N2). To revive a frozen stock, the stock was retrieved from liq N2 and transferred to a beaker containing water at ~37°C. Once the stock has thawed, the cells were immediately transferred to lOmL plain mDMEM and centrifuged at 217 x g for 10 min. The pellet was washed three to four times with plain medium followed by resuspension in mDMEM containing
    15. Preparation and revival of frozen stocks of Leishmania donovani
    16. Murine macrophage cell line J774A.1 (ATCC no. TIB-67) was maintained in phenol red free DMEM supplemented with 10% heat inactivated (45 min at 65°C) foetal bovine serum at 37°C in 5% C02 and 95% air. The cultures were sub-cultured every three days or at the attainment of 80 % confluency.
    17. In vitro J774A.l murine macrophage cultur
    18. Long-term axenic amastigotes were generated by subjecting promastigotes to pH and temperature modulations as described elsewhere (Debrabant et al., 2004). Briefly, live metacyclic promastigotes were harvested by centrifugation and resuspended in DMEM containing 20% FBS and a pH of 5.5 and sub-cultured at 23°C after 72 h three times. Following this, the cells were then transferred to 37°C, 5% C02 for 3 passages after 72 h each. Axenic amastigotes obtained after the last subculture was stained with Giemsa stain and checked under the microscope. They were then maintained at 37°C in a humidified atmosphere containing 5% C02 in air
    19. Leishmania donovani promastigotes (MHOM/IN/80/DD8) were obtained from Dr. R Vishwakarma from the National Institute of Immunology, New Delhi, India. These were grown routinely on blood agar slants containing 1% glucose, 5.2% brain heart infusion agar extract, 6%(v jv) of rabbit blood and 1mg/mL of gentamycin as antibiotic (Sudhandiran and Shaha, 2003) at 23°C. After three days of culture on slants, fresh slants were streaked using a loop for regular maintenance. For liquid cultures, cells were transferred from a slant to modified DMEM (3. 7 g Sodium bicarbonate, 5. 96g HE PES, 5mg Hemin, 1mg Biotin, 13.36mg Adenine, 7.6mg Xanthine, 0.5rnl Triethanolamine, 40mg Tween 80) with 10 % foetal bovine serum (FBS). Before experiments, the cells were centrifuged at 129 x g for 10 min to remove dead and agglutinated parasites; the supernatant was centrifuged at 1258 x g for 10 min to pellet the live cells which were then resuspended in appropriate amounts of media for experiments
    20. In vitro Leishmania donovani culture
    21. Cell Culture Techniques
    22. METHODS
    1. Identification of selected micro-organism
    2. 3.0–5.0, phosphate buffer for pH 6.0–8.0 and Tris-HCl buffer for pH 9.0) were used. •pH stability: The pH stability of the selected tannases was examined in the range of 3.0–9.0 by incubating the enzyme samples for 6 h in different buffers. Tannase activity was estimated under standard assay conditions. •Temperature tolerance: Temperature tolerance of the tannases was examined by assaying their activity at different temperatures in the range of 20 to 80ºC. •Temperature stability: Temperature stability of the tannases was determined by incubating them in the temperature range of 20 to 70 ºC for 6 h. After the incubation tannase activity (%) was determined under standard assay conditions. •Organic solvent stability: In order to determine the suitability of the selected tannases for organic synthesis, their stability was determined in different organic solvents. Experimentally, 10 mg of each of the crude lyophilized tannase from the selected cultures were mixed with 1.0 ml of the following organic solvent: a) Hexane b) Methanol c) Propanol d) Isoamyl alcohol e) Petroleum ether f ) Chloroform The mixture was incubated for 6 h at optimal temperature and the organic solvents were then decanted and the residues were dried in a vacuum desiccator. These dried samples were dissolved in 1.0 ml of citrate phosphate buffer (50 mM, pH 5.0) and the tannase activity was determined under standard assay conditions. The tannase activity thus obtained from each culture were compared with initial tannase activity. Finally, on the basis of tannase titres produced per ml and desirable biochemical properties, the best tannase producer was selected for further investigations
    3. The tannases obtained (at high titres) from selected cultures were evaluated for the following important biochemical properties. 1. pH tolerance and stability 2. Temperature tolerance and stability 3. Organic solvent stability •pH tolerance: pH-tolerance of the selected tannases was examined in the range of 3.0–9.0. Buffers (0.05 M) of different pH (citrate phosphate for pH
    4. Preliminary biochemical characterization of tannases from the potent tannase producers
    5. The reaction mixture contained 10 μl of culture filtrate, 490 μl of double distilled water (DDW) and 300 μl of methanolic rhodanine solution. This mixture was incubated for 5 min at 30°C in a water bath. The reaction was stopped by adding 0.3 ml of methanolic rhodanine solution (0.667 %), which resulted in the formation of complex between gallate and rhodanine. This was followed by the addition of 0.2 ml of KOH solution (0.5N) and the tubes were further incubated at 30°C for 5 min. The total reaction mixture in each tube was diluted with 4.0 ml of distilled water. Tubes were further incubated at 30°C for 10 min. The absorbance was measured at 520 nm against a control having distilled water in place of culture filtrate. The absorbance thus obtained was used to calculate the amount of gallic acid present in the culture filtrate, from the standard gallic acid curve prepared in the range of 100-1000 μg/ml.
    6. The procedure of Sharma et al. (2000) was used to estimate the gallic acid in the culture filtrate. Reagents: Methanolic rhodanine solution (0.667% w/v): Prepared by dissolving 0.667 g of rhodanine in 100 ml of methanol.Potassium hydroxide (0.5 N): 2.8 gpotassium hydroxide dissolved in100 ml of distilled water.
    7. Gallic acid estimation (Sharma et al., 2000)
    8. The tannin sample (1.0 ml) was added to 2.0 ml BSA solution in a 15 ml glass centrifuge tube. The solution was mixed and allowed to stand at room temperature for 15 min and then centrifuged at 10000 rpm for 15 min to separate the precipitated tannin-protein complex as pellet. The supernatant was discarded and the pellet and the walls of the tube were washed with acetate buffer without disturbing the pellet. Now, the pellet was dissolved in 4.0 ml of SDS-triethanolamine solution and to this, 1.0 ml of ferric chloride reagent was added and was mixed immediately. After 30 min of addition of ferric chloride, the absorbance was noted at 510 nm on spectrophotometer. All observations were carried out in triplicates. The concentration of the tannin was determined with the help of tannic acid (Sigma) standard curve prepared in the range of 0.2 to 1.0 mg/ml
    9. The procedure of Hagerman and Butler (1978) was used to estimate the tannin content in different tannin sources. Reagents: Bovine serum albumin (BSA) 1.0 mg/ml: 10.0 mg of bovine serum albumin was dissolved in 10.0 ml of 0.2 M acetate buffer, pH 5.0, containing 0.17 M sodium chloride. Sodium dodecyl sulfate (SDS)-triethanolamine solution: The solution contained 1.0% SDS and 5.0% (v/v) triethanolamine in distilled water. Ferric chloride reagent (0.01 M): 1.62 g of ferric chloride was dissolved in 1.0 L of 0.01 N hydrochloric acid.
    10. Tannin estimation (Hagerman and Butler, 1978)
    11. To 1.0 ml of suitably diluted culture filtrate, 5.0 ml of solution C was added. It was incubated for 10 min at room temperature. To this, 0.5 ml of Folin Ciocalteau’s reagent (diluted 1:1 with distilled water) was added. The solution was vortexed and kept in dark for 30 min. After incubation, absorbance was read at 660 nm against a reagent blank. Protein content was calculated (in mg/ml) using standard curve of bovine serum albumin (BSA) prepared in the range 100-1000 μg/ml
    12. The total protein content in the culture filtrate was estimated by Lowry’s method as described below: Reagents: Solution A: 2.0% Na2CO3 in 0.1 N NaOHSolution B: 0.5 % CuSO4 in 1.0 % Sodium potassium tartarate Solution C: 50.0 ml of solution A was mixed with 1.0 ml of solution B Folin Ciocalteau’s reagent
    13. Protein estimation (Lowry et al., 1951)
    14. For estimation of tannase activity the reaction mixture (4 ml) contained 1.0 ml of 1.0% tannic acid (prepared in citrate-phosphate buffer, pH 5.0), 2.0 ml of citrate-phosphate buffer (pH 5.0) and 1.0 ml of appropriately diluted culture supernatant. The reaction mixture was incubated at 40°C for 30 min in a water bath. The reaction was stopped by adding 4.0 ml of 2.0% BSA solution. In the control reaction, BSA was added prior to incubation. Now the tubes were left for 20 min,at room temperature, for precipitating the residual tannins and subsequently centrifuged at 10,000 rpm for 20 min. The end product, gallic acid thus formed was estimated by diluting 20 μl of the supernatant to 10 ml with DDW. Now, the absorbance at 260 nm was read against a blank (DDW) in a UV spectrophotometer (1601, Shimadzu Corporation, Japan). One unit of tannase: One tannase unit is defined as the amount of enzyme that releases 1 μmol of gallic acid from the substrate (tannic acid) per ml per min under standard assay conditions
    15. In this method, tannase activity was estimated through spectrophotometric method by determining the concentration of the end product i.e., gallic acid, by estimating the absorbance at 260 nm. Reagents: •Tannic acid (1.0%): The solution was prepared by dissolving 1.0 g of tannic acid in 100 ml of citrate-phosphate buffer of the desired pH.•Bovine serum albumin (BSA): BSA (2.0%) was prepared in citrate phosphate buffer (pH 5.0)
    16. Estimation of tannase activity (Deschamp et. al., 1983)
    17. For bacterial isolates, a single colony from a nutrient agar slant was inoculated into 50 ml of nutrient broth in a 250 ml Erlenmeyer flask. These flasks were incubated at 37±1°C in a incubator shaker till an optical density of 0.6 at 660nm. Now these cultures were used to inoculate 50 ml of the tannase production medium in 250 ml Erlenmeyer flasks using 2% v/v inoculum. These flasks were incubated at 37±1°C in an incubator shaker (Multitron AG-27; Switzerland) at 200 rpm for 72h. The experiments were carried out in triplicates. Samples (2.0 ml for bacteria and same for fungi) were withdrawn at regular intervals of 12h upto 72 h. The samples thus obtained were centrifuged at 10,000 rpm in a refrigerated centrifuge (SIGMA 4K15 Germany) for 10 min at 4°C. The supernatant/s were analyzed for tannase activity
    18. For fungal cultures, spores were harvested from 72 hour old cultures grown on PDA/Tannic acid agar slants by adding 10 ml of sterilized normal saline and a few drops of sterilized Tween-80 followed by vortexing. The spore suspension was filtered through sterile cotton filter to ensure that mycelial filaments are removed. The spores were counted using a haemocytometer (Neubaeur). Approximately, 5X106 spores were inoculated in 50 ml of tannase production medium in 250 ml Erlenmeyer flasks. These flasks were then incubated at 30±1 and 37±1°C in an incubator shaker (model G25KC, New Brunswick Scientific, NJ, USA) at 200 rpm
    19. Quantitative assay
    20. was observed by the formation of a clear zone of hydrolysis around the bacterial/fungal colony. Tannase production, in terms of the diameter of the zone of hydrolysis around the colony, was measured (in mm) after 24 (bacteria) and 48 hours (fungi) of incubation. The diameter of the hydrolytic zone was measured at three points and the average was calculated. The microorganisms showing a zone of tannic acid hydrolysis were considered as tannase producers. The potent tannase producers were further tested quantitatively for the amount of enzyme produced in broth.
    21. The procedure of Bradoo et al. (1996), involving point inoculation of the microorganisms on tannic acid agar plates was followed. The plates were incubated at 37 and 30±1°C for bacterial and fungal isolates. The presence of tannase activity
    22. Qualitative screening for tannase producer/s
    23. A total of 150 fungal and 150 bacterial isolates were screened qualitatively and quantitatively for their ability to produce the enzyme, tannase.
    24. Screening and selection of potential tannase producers
    25. Microorganisms were isolated from the above mentioned sources using direct plating method. Serial dilution of the different soil samples with normal saline was carried out and the different dilutions were spread plated on to potato dextrose agar (PDA) for isolation of fungi and on to nutrient agar (NA) for the isolation of bacteria. The plates were incubated at either 30 or 37±1°C in a bacteriological incubator so that the different organisms could grow and form visible colonies. The different fungal and bacterial colonies isolated by the procedure mentioned above were purified by subculturing on respective media, and subsequently screened for tannase production. The new isolates, alongwith different cultures obtained from laboratory stock culture collection, were revived on potato dextrose agar (PDA) slants. These cultures were regularly subcultured and stored at 8±1°C in a BOD incubator. Their purity was periodically checked by microscopic examination.
    26. Isolation of bacteria and fungi from the samples
    27. In the present investigation, microorganisms including both bacteria and fungi were isolated from soil samples collected from different geographical locations in India. Microorganisms were also isolated from the bark of trees as well as from the soil near the roots of those trees. Some cultures were also procured from the laboratory stock culture collection.
    28. Collection of samples
    29. Isolation of tannase producing microorganism/s
    1. Cultures in mid-exponential phasenormalized using A600and solubilizedin 1X sample buffer at 99°C for 5 min were subjected to electrophoresis on 12% sodium dodecyl sulfate (SDS) -polyacrylamide gels. Cell extracts equivalent to 0.04A600(1X) and 0.02A600(0.5X) were loaded and run using Tris-glycine-(SDS) buffer. Separated proteins were electrotransferred to PVDF polyvinyledene difluoride) membrane (Amersham) electrophoretically by a semi-dry method using Bio-Rad apparatus.The transfer was done for 2-3 hrs using a voltage of 75V at 4oC and membrane was probed using anti-FtsZ primary antibody at 1:5000 dilution (rabbit, polyclonal), washed and probed with anti-rabbit IgG conjugated to horseradish peroxidase (HRP) at 1:20000 dilution, as described(Sambrook & Russell, 2001).Membranes were developed with chemiluminescencereagent (Amersham ECL Prime) and visualized with the aid of a chemiluminescence detection system according to the manufacturer’s protocol (Sigma Chemical Co., St. Louis, MO). Quantification of band intensity and subtraction of background was done using Fujifilm Multi Gauge V3.0 imaging system(Image Quant software)
    2. Gel Electrophoresis and Western blotting
    3. Viewing slides under microscope
    4. A drop of immersion oil was put on top of the cover-slip before viewing it under microscope. The cells were viewed at 100X resolution of Nikon Eclipse 80i microscope.Thedifferential interference contrast images of the cells were captured using NIS-Elements D3.0 software also used to find out mean cell size using at least 100 randomly selected cells.Fluorescence images were captured on Zeiss LSM 710 Meta inverted confocal microscope
    5. The slides for microscopy were prepared as described in Dajkovic et al.,(2008)with slight modifications. After wiping the glass slide with ethanol, 200μL of 1% molten agarose was layered on it between two strips of tape and clean cover-slip placed on it to obtain levelled surface. The agarose was allowed to solidify and the cover-slip was carefully removed and5μlof sample was put on top of the agarose and carefully covered with a cover-slip
    6. Preparation of microscopic slides
    7. Fresh overnight cultures grown in LB containing appropriate antibiotics to select for plasmids were sub-cultured 1:100(or lower dilutions for some strains)in the same medium. The cells from these cultures weretaken for microscopy at exponential phase of growth(A600 of 0.5-0.6), as such or after concentrating the cells 10-fold
    8. Sample preparation
    9. Microscopy
    10. The method followed was similar to that describedpreviously with slight modifications (Jinet al., 1992; Schleifet al., 1973).Overnightbacterial cultures were grown in LBand subcultured 1:500 in the same medium in a volume of 20 mlat 30oC.Cultures were induced with 1mM IPTG at A600=0.4. 0.9ml samples were aliquotedat time intervals of 0 sec, 20 sec, 40 sec, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min, 5 min, 5.5 min and 6 min into 0.1ml of 1mg/ml ice cold chloramphenicol and the samples were put on ice. After sampling, 0.5ml of each culture was taken for β-galactosidase assay.Square root of β-galactosidase activity (activity at time Tt−T0) was plotted against time. In the graph, the point of inflection of the curve on the X-axis determines the rate of elongation of RNAP whereas slope represents the promoter clearance, lacZmRNA stability and factors affecting translation of lacZ(Burovaet al., 1995)
    11. RNA polymerase elongation rate measurement
    12. The method followed was as described in Miller (1992). Samples for dot-blotting were prepared by mixing 5μg of RNA (in10μl H20) with 30μl of RNA denaturing solution consisting of 1X MOPS, 7% formaldehyde and 50% deionised formamide. The samples were heat denatured at 65oC for 5 min and mixed with equal volume of 20X SSC. The samples wereloaded into the slots of the dot-blot apparatus (Bio-Rad) containing the membrane (pre-soaked in 20X SSC)and gentle suction was appliedusing Millipore vacuum pump. The slots were rinsed twice with 10X SSC. This was followed by the UV-crosslinking, pre-hybridization, hybridization, washing and exposure of the membrane identical to that done in Northern blotting
    13. Dot-blotting
    14. Non-stringent washes were carried out in 2XSSC and 0.25-0.5% SDS in DEPC water.Stringent washing was done in 1XSSC and 0.5% SDS in DEPC water. Washing was carried out at 55-56oC for 20 minutes. After washing, the blot was covered in the saran-wrap and exposed to the phosphoimager film. After the desired time of exposure, the filmwas then scanned in phosphoimager and the picture saved.The densitometric analysis of the bands was carried out as described in the section of the signal intensities in northern blotting experiments using probe against tRNA(U73)Arg5was done as follows. The intensity of the tRNA(U73)Arg5signalin the WT or the parent strain in the absence of IPTG was taken as 1 and the relative change in the other strain/growth condition calculated. The value thus obtained was corrected using the change in the corresponding 5S rRNA intensity relative to that in the WT/parent strain in the absence of IPTG
    15. Washingof the membrane, exposure and scanning
    16. For hybridisation,probe was heated at 95oC for 5 minutes and snap-chilled for 5 minutes and then added to the hybridisation bottles containing the blot. Hybridisation was carried out overnight at 50oC.The probes used and their radioactivity counts (in parentheses) were 5s RNA probe (2.5×106cpm), U73 probe (5×106cpm) and lacZ probes (106 cpm)
    17. Hybridisation of the membrane
    18. ii.10% Dextran Sulphateiv.0.5% SDSv.100 μg/mlSalmon Sperm DNAvi.DEPC water5ml of pre-hybridisation buffer was used per blotin 150×35mmhybridisation bottles(Labnet). Salmon sperm DNA was heated at 95oC for 5 minutes and snap-chilled for 5 minutes prior to adding to the rest of the mix. Blot was inserted into the bottle such that it stuck to the wallsand the surface containing the RNA faced the inner side of bottle. Pre-hybridisation was carried out at 50oC for 3 hours in hybridisation chamber(Labnet Problot 12S hybridisation oven)
    19. The pre-hybridization buffer contained the following constituents:i.6X SSCii.5X Denhardt’s solution
    20. Pre-hybridization of the membrane
    21. The RNA was cross-linked onto the membrane after transfer by exposing it to the UV light of 200KJ/cm2 energy in a UV-crosslinker
    22. UV-crosslinking of the RNA
    23. Semi-dry transfer apparatus (Bio-Rad trans blot semi dry transfer cell)was used for the transfer of RNA from the gel to the membrane. The Hybond-N+ membrane from Amersham biosciences was used which was cut as per dimensions of the gel containing the RNA samples. For each transfer 6 pieces of Whatman3mmsheets of the size of the membrane were used. The membrane was soaked for 30-60 minutes in 0.5XTBE before transfer. The transferapparatus was set up as describedby the manufacturer. Transfer was done in 0.5XTBE buffer at 20V, 400mA and 100W for 1.15 hours
    24. Transfer of RNA to the membrane
    25. vii.RNA buffer II from Ambion(1-2X Xylene cyanol + Bromophenol blue)used for loading the samples. RNA isolation for Northern blotting for lacZtranscript was done aftergrowing cultures till A600of 0.6 in LB in the presence or absence of 1mM IPTG at 30oC while for lacZ-lacYʹ-tRNA(U73)Arg5or lacZʹ-tRNA(U73)Arg5transcripts, cultures were grown in LBupto A600of 0.3 and induced with 1mM IPTG for 30 min followed by RNA extraction.30ml of 10% polyacrylamide gels of 1.5mm thickness were cast in the Broviga slab vertical gel electrophoresis apparatus. Gels were polymerizedby the addition of TEMED and APS (1/100th volume of gel mix). The gel was pre-runat 300V for 15-20 minutes prior to loading.Sample preparation for gel loading was done as follows. The normalizedamounts of RNA samplesto be analyzed were mixed with the equal volumes of 2X gel loadingbuffer II(Ambion)making a final concentration of 1X. The samples were then heated at 80 degrees in a thermoblock (eppendorf) for 10 minutes and loaded on the gel when still warm. The gel was run at constant voltage of 300Vfor 3-4 hours till xylene cynol covered 2/3rddistance
    26. The following solutions were used to cast and run denaturing PAGE gels:i.40% acrylamidestock solution ii.7.5M Ureaiii.5X TBEiv.Ammonium persulphate (APS) stock: 10% (w/v) solution made fresh v.TEMED (N,N,N′, N′-tetramethyl ethylene diamine) vi.Gel running buffer (0.5X TBE)
    27. Denaturing polyacrylamide gel electrophoresis of RNA
    28. The method followed was as described in(Lopezet al., 1997)with few modifications. The steps are as described
    29. Northern Blotting
    30. Band intensities in gel autoradiogramswere determined by densitometry with the aid of the Fujifilm Multi Gauge V3.0 imaging system. Equal areas of radioactive bands were boxed and the PSL (Photo stimulated luminescence) values were further considered. Background signal (obtained from equal area as that of the radioactive band but from other part of the gel/blot) is subtracted from the signal intensities obtained from radioactive bands to get the final values
    31. Densitometry
    32. Oligonucleotides and PCR products were end-labelled using phage T4-polynucleotidekinase (PNK, New England Biolabs or Fermentas or Sigma) with 32P-γ-ATP. The radiolabelling reaction mixture (20μl) contained 1X of buffer provided by the company, 10 units of T4-PNK and 40μCi of 32P-γ-ATP. The reaction mix was incubated for 1 hrat 37ºC and the reaction was heat-inactivated at 65oC for 20 minutes. The labelled oligonucleotides and DNA fragments were purifiedby the Qiagen nucleotide removal kit. Labelling efficiency was checkedeither by using Geiger-Muller (GM) counter orusing liquid scintillation counter.For scintillation counting, 1μl of radioactive sample wasadded to the 5ml scintillation cocktail, and radioactivity count was determined in the 32P channel of scintillation counter (Perkin Elmer, Liquid Scintillation analyzer, Tri-Carb 2910 TR, USA). Liquid scintillation cocktail consists of 5g PPO (2,5-diphenyloxazol) and 0.3g POPOP (1,4-bis (5 phenyl 1,2-oxazole) Benzene, adjusted to a volume of 1L in toluene
    33. Radiolabelling of oligonucleotides
    34. DNA sequencing
    35. Automated DNA sequencing on plasmid templates or on PCR products was carried out with dye terminator cycle sequencing kits on an automated sequencer following the manufacturer's instructions byan outsourced sequencing facility
    36. The semi-quantitative reverse transcription-PCR (RT-PCR) involves the synthesis ofcomplementary DNA (cDNA)from RNA. For this, 1μgof RNAwas treated with 1μl (1 unit) DNase I enzyme (Sigma, amplification grade) for20 min to remove DNA contamination. DNase I was inactivated by heating at 70oC for 10 min. Next, 5pmol reverse primer wasadded along with dNTPs and volume made to 10μlwith DEPC-treated water; the mix washeated at 65oC for 5 min and incubated on ice forat least 1 min. The reverse transcription reaction was set up with this mix using the Superscript III RT kit (Invitrogen) as per manufacturer’s protocolto obtain cDNA. The cDNA servedas the template for setting up a PCR for requirednumber of cycles. The samples were finally run on agarose gels
    37. Reverse transcription(RT)-PCR
    38. Concentrations of DNApreparations were estimated by nanodrop or by gel electrophoresis followed by densitometric analysis.Concentration of RNA preparations were estimated by nanodrop
    39. Estimation of DNA and RNA concentrations
    40. Total RNA extraction from E. colicells was doneusing Qiagen RNeasy minikit. Cells were grown to an A600of 0.6 and harvested(amaximumof107cells)at 6000rpm for 5min at room temperature to prevent cells for encountering any stress in cold. Rest of the steps were followed exactly as mentioned in the manufacturer’s protocol. The quality of RNA preparations was assessed following electrophoresison 1.4% agarose-formaldehyde-MOPS gels.Ingeneral,forawild-typestrainRNAyieldwouldbe~0.5-1μg
    41. Isolation of total cellular RNA
    42. For high fidelity PCR, Herculase II fusion DNA polymerase (AgilentTechnologies)was used. Approximately 0.5μg of chromosomal DNAwas used as a template in a 50μl reaction volume
    43. The PCRs were normally performed using Taqpolymerasefrom Roche or Fermentas. Approximately 1-5ng of plasmid or 5-100ng of chromosomal DNA was used as a template in a50μlreaction volume containing 200μM of each dNTP, 20pM each of the forward and reverse primers and 1 unit of Taq DNA polymerase. For colony PCR E. coli cells from a freshly grown plate were resuspended in 10μl of sterile Milli-Q water to get a cell suspension and this was used as a template in a PCR reaction at a final volume of 50μl. The samples were typically subjected to 30 cycles of amplification with the following general conditions: Initial denaturation 95ºC5minutes Denaturation 95ºC 1 minute Annealing 55ºC 1 minute Extension 72ºC 1 minute/kb of DNA template to be amplified Final extension 72ºC 10 minutes
    44. Polymerase Chain Reaction (PCR)
    45. Molecular techniques
    46. Recombineering was performed as described in(Yuet al., 2000)for engineering the linear DNA on the chromosome. The oligonucleotide primers were designed to amplify the DNA cassette to be engineered. Oligonucleotidesused for recombination contained30–50nt homology at the 5ʹ endtothesequences at the target siteand 20nt homology tothe DNA cassette at the 3ʹ end. The DNA cassettefor recombinationwas generated by PCR and would contain30-50 bp homologiesto the target site. A strain with the target DNA and carrying a defective λ-prophage with gam,betaand exo genes (thatfacilitate homologous recombination)under the control of a temperature-sensitive λ cI-repressorwas grown at 30oC. At an A600of 0.4, the culture was shifted to 42oC for 15 minutes to express gam,betaand exo genes. Cells becomecapable ofrecombining linear DNA introduced into the cell by electroporation. 50-100ng ofamplified DNA cassettewas used for electroporation whichwas performed using theBio-Rad Gene Pulser set at 1.8 kV, 25 μF with Pulse controller of 200 ohms
    47. Recombineering
    48. Typically 400-500ng of DNA was used in each ligation reaction. The ratio of vectorto insert was maintained between 1:3 and 1:5 for cohesive end ligation. The reaction was generally performed in 15μl volume containing ligation buffer (provided by the manufacturer) and 0.075 Weiss unit of T4 DNA ligase at 16ºC overnight (14-16 hours)
    49. Ligation of DNA
    50. DNA fragments to be used for specific purposes like ligation or radioactive labellingwere eluted from the agarose gel after electrophoresis. The gel piece containing the desired band was sliced out from the gel and the DNA was purified using commercially available purification kit (Qiagen)for this purpose. The efficiency of elution was determined by checking a small aliquot of DNA sample on the gel
    51. Purification of DNA by gel elution
    52. Around 0.5-1μg DNA was regularly used for each restriction digestion. 2 to 5 units of restriction enzyme were used in the total reaction volume of 20μl containing 2μl of the corresponding buffer supplied at 10X concentration by the manufacturer. The reaction was incubated for 3hours at the temperaturerecommended by the manufacturer. The DNA fragments were visualized after electrophoresis on 0.8 to 1.5% agarose gels. Commercially available DNA size markers were run along with the digestion samples to compare with and to estimate the sizes of the restriction fragments
    53. Restriction enzyme digestion and analysis
    54. The DNA samples were mixed with appropriate volumes of 6X loading dye (0.25%bromophenol blue and 0.25% xylene cyanol and 30% glycerol in water) and subjected to electrophoresis through 0.8 to 1.5 % agarose gel in TAE buffer. The Goodview nucleic acid stain(supplied as 20000X; Beijing SBS Genetech Co. Ltd.) was added to the gel at the time of casting or 6X EZ-Vision One DNA dye(Amresco) was used as loading buffer, both being commercially available non-carcinogenic dyes to aid visualization of bands. The visualization was doneby fluorescence under UV light in a UV transilluminator
    55. Agarose gel electrophoresis
    56. following the manufacturer’s instructions. For genomic DNA, 1ml culture was used for DNA isolationusing Qiagen or Invitrogen kits. The quality of plasmid/genomic DNApreparations was assessed following electrophoresis on 0.8% agarose gels
    57. 3ml (for high copy number)or 10 ml (for low-copy number) of cells from an overnight culture were pelleted by centrifuging for 5 minutes at 6000rpm forthe plasmid isolation which was carried out with the commercially available kits (Qiagen or Invitrogen)
    58. Isolation of plasmid and chromosomal DNA
    59. Recombinant DNA techniques
    60. Growth curves were generated to compare the growth rates of E. coli test strains with control strains manually. The appropriate dilutions of the overnight cultures in desired media were made and allowed to grow at required temperature till faint turbidity was visible. At this point samples were collected every 30 minutes until stationary phase was attained. The growth curves weregenerated using Microsoft Excel or SigmaPlot software and growth rates were calculated from the slope of the graph which, in turn, was used to calculate generation time
    61. Estimation of growth rates
    62. β-Galactosidase assay was performed according to(Miller, 1992).Cultures were grown to A600 of 0.4-0.6 from a 1:100 dilution of overnight cultures. Around 0.1-0.5 ml of culture was made up to 1 ml with Z-buffer and lysed with the addition of 100μl of chloroform and 50μl of 0.01% SDS solution. 0.2ml of freshly prepared 4mg/ml ONPG was added to start the reaction and incubated at 28oCtill the colour of the reaction mixture turned yellow. 0.5ml of 1M Na2CO3 was added to stop the reaction and the time duration from initial addition of ONPG to the stopping of the reaction was noted. The absorbance of reaction mix was taken at 420 nm (A420) afterspinning down the mix at 12000rpm for 3 minutes. The A600of the culturesused was also noted. The enzyme’sspecific activity (in Miller units) was calculated using the following equation: β-Galactosidase specific activity (Miller units) = (1000 ×A420) / t × v ×A600Where,‘t’ is the time period in minutes and ‘v’, the volume of culture used in ml
    63. white colonies were recovered and purified to give growth. If the mutation caused synthetic lethality then white colonies (that lack the shelter plasmid) would not be observed since plasmid loss would result in growth arrest. Therefore, lethality was inferred when either white colonies were not recoveredor were recovered but failed to purify further
    64. To determine whether a particular mutation conferred lethality in the ppGpp0or ΔdksAbackground, an assay was devised based on the use of an unstable, easy to cure shelter plasmidpRC7, similar to that described previously(Bernhardt & de Boer, 2004). In the wild-type strain carrying pRC7, this plasmid can be lost at a frequency of 20-30% in the absence of the selection. However, this will not be seen if the plasmid loss leads to cell death. Since the plasmid pRC7 confers a lac+phenotype, in the absence of the selection plasmid loss can be visualized on X-gal IPTG containing plates as white colonies in a Δlac strain whereas the colonies that retain the plasmid will appear blue.In order to carry outsynthetic lethal screen in the ppGpp0or ΔdksAstrains, the spoT or dksAgenes cloned in pRC7 under the control of lacpromoter were used. Theseshelter plasmids,namely,pRCspoT or pRCdksA, respectivelywere transformed into the ppGpp0or ΔdksAstrain. To test the synthetic growth phenotypes, the mutations of the genes to be tested were introduced by phageP1 transductions. The resultingstrains were grown overnight in LBcontaining the antibiotic selection for the shelter plasmid and IPTG for expression of spoTor dksA, subsequently washedin minimal A medium and dilutions(usually 10−5or 10−6) of these cultureswere spreadon X-gal and IPTG containing plates without antibiotic selection for the shelter plasmid. The phenotypes of the white colonies in comparison with the blue colonies were noted. Viability of the strains was inferred when
    65. Blue-white screening for viability or lethality phenotype
    66. or absence of a metabolite or a particular temperature. An EOP of ≤0.01 suggests lethality of the strain on the test medium. For strains carrying IPTG-dependent plasmids, EOP was determined by growing the strains overnight in medium containing IPTG and appropriate antibiotic,and spottingserial dilutions (100or 10–1to 10–6) on +IPTG (permissive) and –IPTG (test) plates to observe growth. Theviability is scored by takingratio of the colony forming units per ml (cfu/ml = No. of colonies × dilution factor × 1000/volume of culture spotted (in μl) obtained on the –IPTG plate to that on the +IPTG plate and determinesthe EOP. Likewise, strains carrying Ts plasmids were cultured overnight at 30°C with the appropriate antibioticand the serial dilutions of this culture were spottedat two temperatures 30°C (permissive) and 42°C (non-permissive or test). The ratio of cfu/mlobtained on the test temperature to that on the permissive temperature determined the efficiency of plating at the test temperature
    67. Efficiency of plating (EOP) is a measure of the ratio of number of colonies (obtained from a given volume of a suitable culture dilution) on a test medium to those on a control or permissive medium, and is a measure of cell viability on the former. It is a very sensitive test and is often used for determining the viability of a strain in the presence
    68. Efficiency of plating (EOP)
    69. C. LBON temperature-sensitivityStrains were streaked on LBON agar plates and after an overnight incubation at42°C, growth was monitored (compared to that on LBON at 30°C as control). Absence of single colony growth was taken to reflect temperature sensitivity. D. In vivotranscription termination phenotypes The rationale for each phenotype is described in the relevant section. SMG-sensitivityThe E. coli relA mutants exhibit SMG-sensitive (SMGs) phenotype,that is,growth-inhibition in the presence of serine, methionine and glycine at 1mM concentration each(Uzan & Danchin, 1978)and is proposed to be a consequence of transcriptional polarity exerted by a frameshift mutation in the ilvG gene on the expression of downstream genes of the ilvGMEDA operon(Lopes & Lawther, 1989).This test was therefore used to distinguish relA+from relA−strains. Growth in the presence of amino acids serine, methionine, and glycine (SMG) was scored on glucose-minimal A plates supplemented with each of the amino acids at 40μg/ml and compared with the growth on non-supplemented glucose-minimal A plates to score for SMG phenotype. galEp3assayThis assay was used to test for relief of transcriptional polarity in the rho and nusG mutants. The galEp3 (galE490*) mutation represents a 1.3kb IS2 insertion in the gal leader region (between the promoter and structural genes of the galETKM operon). The mutation causes transcriptional polarity on the structural genes due to Rho-dependent transcription termination within IS2. In this assay, the gal operon expression in a galEp3 mutant or its derivatives was monitored by usingMacConkey galactose indicator plates (with 1% galactose), where Gal+colonies are red, and Gal−colonies are white. Therefore, the depth of color serves as an indicator of relative levels of gal expression, i.e., the extent of transcriptional polarity relief
    70. A.lacZphenotype lacZ+colonies were distinguished from lacZ–colonies on X-gal containing plate or MacConkey lactose plate. X-gal is non-inducing colourless substrate of β-galactosidase enzyme which upon hydrolysis yields dark blue indolyl group and hence the lacZ+colonies on X-gal plate appear as dark blue colonies. Similarly, on the MacConkey agar plateslacZ+colonies appear dark pink whereas lacZ–colonies remain colourless. B. UV-sensitivityTo check the UV-sensitivity of the strains qualitatively, the strains were streaked on duplicate LB-agar plates and one of the plates was UV-irradiatedwith a 15-W UV-germicidal lamp at a distance of 70cm for 30 seconds. The UV-exposed and unexposed plates were incubated overnight in the dark after wrapping with aluminium foil and then growth was scored. This test could differentiate a recA–strain (UVs) from a recA+strain (UVr)
    71. Scoring for Phenotypes
    72. desired temperaturefor 45 minutes and plated on an appropriate selective medium at various dilutions. An aliquot of cell suspension to which plasmid DNA was not added served as a negative control. B. Inoue method i. Preparation of high efficiency competent cells Competent cells for high efficiency transformation were prepared by the method of (Inoueet al., 1990)with few modifications. An overnight culture of the strain (routinely DH5α) was subculturedinto fresh sterile LB broth in 1:100 dilutions and grown at 18ºC to anA600of 0.55. The cells were harvested by centrifugation at 2500rpm for 10 minutes at4ºC. Thesecells wereresuspended in0.4 volumes of INOUE buffer andincubated in ice for 10 minutes. The cells were recovered by centrifugation at 2500rpm at 4ºC for 10 minutes and finally resuspended in 0.01 volume of the same buffer. Sterile DMSO was added to a final concentration of 7%. After incubating for 10 minutes in ice, the cells were aliquoted in 100μl volumes, snap frozen in liquid nitrogen and stored at –80ºC. ii. Transformation protocolFor transformation, the required number of vials wasthawed on ice and the transformation protocol as described for CaCl2method was employed
    73. A. Calcium chloride(CaCl2)method For routine plasmid transformation, following method which is a modification of that described by(Cohenet al.,1972)was used. An overnight culture of recipient strain was subcultured 1:100 in fresh LB medium and grown till mid-exponential phase. The culture was chilled on ice for 20 minutes, and the steps thereafter performed at 4ºC. 10 ml of culture was centrifuged and pelletwas resuspended in 5 ml of 0.1M CaCl2. After 5 minutes of incubation on ice, the cells were again centrifuged and resuspended in 1ml of 0.1M CaCl2. The suspension was incubated onice for 45 minutes. To the 100μl aliquot of the cellsuspension plasmid DNA (20-200ng in less than 10μl volumes) was added, incubated for 30-40 minutes on ice and given a heat shock for 90 seconds at 42ºC. The cultures were rapidly chilled for 1 minute, mixed with 0.9ml of LB broth and incubated at
    74. Transformation
    75. To 2 ml of fresh overnight culture of recipient strain, 108 pfu equivalent of phage lysate was added and incubated at 37ºC without shaking for 30 minutes to facilitate phage adsorption. The unadsorbed phage particles were removed by centrifugation at 6000 rpm for 5 minutes and the pellet ofbacterial cells was resuspended in 5 ml of LB broth containing 20 mM sodium citrate to prevent further phage adsorption. This was incubated for 25-60 minutes at desired temperaturewithout shaking to allow the phenotypic expression of the antibiotic resistance gene. The mixture was then centrifuged and the pellet was resuspended in 300 μl of 0.1M citrate buffer. 100 μl aliquots were spreadon appropriate antibiotic containing plates supplemented with 2.5 mM sodium citrate. A control tube without addition of P1 lysate was also processed in the same way. In the case of selection of nutritional requirement, the infection mixture was centrifuged, resuspended in 300 μl of 0.1M citrate buffer and plated without phenotypic expression
    76. 10–6)were mixed with 0.1 ml of fresh culture grown in Z-broth. After 30 min of adsorption at 37ºC without shaking, each mixture was added on a soft agar overlay of Z-agar plates and incubated overnight at 37ºC. The phage titrewas calculated from the number of plaques obtained on the plates as follows: Phage titre(pfu) per ml = No. of plaques ×dilution factor ×1000/vol.of lysate added (in μl)
    77. Phage P1 lysate preparation by broth method
    78. Phage P1 transduction
    79. 0.3 ml of overnight culture of the donor strain in Z-broth was mixed with 107plaque forming units (pfu) of a stock P1 phage lysate prepared on strain MG1655. Adsorption was allowed to occur at 37ºC for 30 minutes and the lysate was prepared by broth method. To 0.3 ml of infection mixture, 8-10 ml of Z-broth was added and incubated at 37ºC with slow shakinguntil the visible lysis of the culture occurred (in 4-6 hours). The lysate was treated with 0.2ml of chloroform, centrifuged and the clear lysate wasstored at 4ºC with chloroform.Quantitation of Plaque forming units (pfu)To quantitate the titreof P1 lysate preparation, titration was done using P1 phage sensitive indicator strain such as MG1655. 100 μleach of dilution of phage (typically 10–5,
    80. Genetic Techniques




  2. Apr 2019
    1. But thirdly, and most valuably, the template gives you a big space at the bottom to write sentences that summarise the page.  That is, you start writing your critical response on the notes themselves.

      I do much this same thing, however, I'm typically doing it using Hypothes.is to annotate and highlight. These pieces go back to my own website where I can keep, categorize, and even later search them. If I like, I'll often do these sorts of summaries on related posts themselves (usually before I post them publicly if that's something I'm planning on doing for a particular piece.)

  3. Mar 2019
    1. We performed some manipulation checks to examine the internal validity of the perceptual-cognitive skill tests and any learning effects as a result of watching the same video clips multiple times
    1. Mager's tips on instructional objectives This is a very simple page that consists of black and white text without any graphics. As is, the text on the page is rather small and difficult (for me, anyway) to read, so one may wish to enlarge it. The process of creating instructional objectives in this format is explained in a clear and straightforward way. Rating 5/5

  4. Oct 2018
    1. Four groups of subjects were compared in the study.
      1. offenders of violent criminal activity: impulsive, non-deliberate, affectively motivated, affectively aggressive

      2. control group: no criminal activity and no mental disorder

      3. violently deliberating behaving delinquents: non-impulsive

      4. delinquents performing property criminal activities, non-violent, non-impulsive

    1. (1)studies relating clinical focal frontal lobe disor-ders to violent behaviour; (2) studies reportingneuropsychological measures of frontal lobefunction in aggressive and antisocial subjects;(3) studies of clinical neurological findings inviolent and criminal populations; and (4) neu-roimaging studies of aggressive and violentsubjects

      (2) and (4) are empirical research

    2. Methods

      critical review; compiled information from research articles and studies is used to provide evidence for the connection between frontal lobe dysfunction and violent/aggressive behavior

    1. Second, we recruited a group of college students majoring in chemistry tocompletetheprocedure
    2. Weemployed two control groups as a performance standard. First, as in our pre-vious study, we asked a group of people to fill out the questionnaire withoutproviding them any information whatsoever about the specific case except itstype (i.e., murder).
    3. First, a group of senior police detectives who possessedsubstantial experience in criminal investigations participated. Second, werecruited a group of detectives who specialized in the investigation of homi-cide. Third, to contrast investigative experience to general experience as apolice officer, a sample of trainee detectives who possessed significant expe-rience in police general duties but only a marginal amount of experience incriminal investigations was obtained. Finally, a sample of police recruits wasalso used.
  5. Sep 2018
  6. www-jstor-org.proxy.library.georgetown.edu www-jstor-org.proxy.library.georgetown.edu
    1. Our research took place at Florida State University (FSU), a large, flag-ship, Research–1 institution in the Southeast US, had IRB approval and spanned two semesters totaling thirty-five weeks, over the fall of 2009 and the spring of 2010.
  7. Aug 2018
    1. Weber notes that according to any economic theory that posited man as a rational profit-maximizer, raising the piece-work rate should increase labor productivity. But in fact, in many traditional peasant communities, raising the piece-work rate actually had the opposite effect of lowering labor productivity: at the higher rate, a peasant accustomed to earning two and one-half marks per day found he could earn the same amount by working less, and did so because he valued leisure more than income. The choices of leisure over income, or of the militaristic life of the Spartan hoplite over the wealth of the Athenian trader, or even the ascetic life of the early capitalist entrepreneur over that of a traditional leisured aristocrat, cannot possibly be explained by the impersonal working of material forces,

      Science could learn something from this. Science is too far focused on the idealized positive outcomes that it isn't paying attention to the negative outcomes and using that to better define its outline or overall shape. We need to define a scientific opportunity cost and apply it to the negative side of research to better understand and define what we're searching for.

      Of course, how can we define a new scientific method (or amend/extend it) to better take into account negative results--particularly in an age when so many results aren't even reproducible?

  8. Jul 2018
    1. I buy into Newton’s philosophy that we see further by standing on the shoulders of giants.

      I take his general point here, and Newton said something along these lines, but I wouldn't call it "Newton's philosophy". If anything this philosophy is really the scientific method and Newton didn't invent it.

    1. Luhmann didn’t only write a lot and developed the most complex of all theoretical bodies in the social sciences. He was known for his vast knowledge and deep thinking. He didn’t run to his Zettelkasten when you asked him something. This is because he practiced thinking through writing and processing in the context of the Zettelkasten.

      I read Zettelkasten (German for “slip box”, or “card index”) and immediately think commonplace book!

    2. The Barbell Method takes this into account by integrating your reading habit into your knowledge work with two steps: Read the book. Read swiftly but don’t skip any parts unless they make you vomit or put you to sleep. Mark all the passages that stand out and contain useful, interesting or inspiring information. Read the book a second time. But now you read the marked parts only. This time you make notes, connect them to past notes (Zettelkasten Method!) and think about what you’ve read. Make mindmaps, drawings, bullet points – everything that helps you to think more clearly.
  9. May 2018
    1. Instead of being simply the recipient of negative comments, the reader, in this situation, sees an alternative possibility.  It's not the only one.  It's another option on the way to success. 

      love it

  10. Mar 2018
  11. Nov 2017
    1. A practice is included in our list if large numbers of researchers use it and large numbers of people are still using it months after first trying it out. We include the second criterion because there is no point in recommending something that people won't actually adopt.

      I like the criteria for inclusion (and the whole article), but one could wonder if they really makes something a "good enough" practice. How many is "large numbers of researchers"?

  12. Oct 2017
    1. applying networkand content analyses

      I came across this article while doing research for last week’s blog. I know this is not a straight forward SNA article, but I found it very interesting since it is a combination of SNA and content analysis. Considering this week’s readings on different data collection method, I found their approach of collecting data from Twitter very unique. In this context, content analysis refers to analyzing tweets and their content. Recently, content analysis is being used in various fields. Even social researchers are taking this opportunity of exploring already existing data. Do you think you can use the combination of both SNA and content analysis in your own research field?

  13. Jul 2017
    1. I have written elsewhere: ‘The way of becoming actually free is both simple and practical. One starts by dismantling the sense of identity that has been overlaid, from birth onward, over the innate self until one is virtually free from all the social mores and psittacisms. Virtually free from all the beliefs, ideas, values, theories, truths, customs, traditions, ideals, superstitions ... and all the other schemes and dreams. One can become aware of all the socialisation, of all the conditioning, of all the programming, of all the methods and techniques that were used to produce what one thinks and feels oneself to be – a wayward identity careering around in confusion and illusion. A ‘mature adult’ is actually a lost, lonely, frightened and very cunning entity. However, it is never too late to start in on uncovering and discovering what one actually is. One can become virtually free from all the insidious feelings – the emotions and passions – that fuel the mind and give credence to all the illusions and delusions and fantasies and hallucinations that masquerade as visions of The Truth. One can become virtually free of all that which has encumbered humans with misery and despair and live in a state of virtual freedom ... which is beyond normal human expectations anyway. Then, and only then, can the day of destiny dawn wherein one becomes actually free. One will have obtained release from one’s fate and achieved one’s birthright ... and the world will be all the better for it’.
  14. Apr 2017
    1. This article can be utilized as an argument piece wherein I can help build on my argument.

      Empty rhetoric here. Can cut it. It doesn't say anything really, right?

  15. Feb 2017
    1. mechanistic approach

      "ars est celare artem: art consists in concealing art"

      I do not dig this mechanical, technical, scientific method dissection of writing. Unfortunately, this article is filled with this pre-Freudian crap. You wouldn't tear Raphael a new one because he painted The School of Athens figures in the wrong order.

      Are these mechanics the result of the scientific method?

  16. Dec 2016
    1. results of your research, and within the context of the study, we found ample evidence about significant cha

      You can get it down even more, no? "Your research suggests we should make . . . "

  17. Oct 2016
  18. Sep 2016
  19. Aug 2016
    1. Performance CategoryDesign Categoriesi. StructureFrame design, shape and materials –for functionii. MobilityThrusters: number, power, orientationiii. SensorsCameras, lights, sonar, touch sensors, compass, GPSiv. ToolsArms, claws, rakes, wrenches, hammersv. Ranging DistanceTether length: waterproofing required vi. Buoyancy/ BallastFixed or variable, location and materialsvii. ControlsRC via wire or signal via fibre optic cableviii. Other?Depends on the specific mission

      Are you doing science projects? Maybe you can use an old mission scope to have students ask questions about. That way some of the questions we will need to face will be answered before we actually get the mission for this year.

    1. For the humanities syllabi, I also asked how many tools were being taught in each course.
    2. I looked at whether the courses: 1) required a collaborative project and 2) set aside time to discuss the challenges of collaboration or cross-disciplinary research (or had readings that indicated such).
    3. With each syllabus I looked for general focus (is it tools-, training-, or topics-focused?), the breadth of assigned readings (is the literature from librarianship, humanities fields, or both?), and the structure of project(s) (collaborative, individual).
  20. Jul 2016
    1. Page 15

      Rockwell and Sinclair on the importance of staying up-to-date on commercial developments in text mining and text-handling:

      we are practicing thinking in the humanities while the way people read, the tools of reading, and information privacy and organization are shifting around us. These shifts matter. If we continue to treat textuality as a subject, we need to understand how text can be mined.

    2. Page 6

      Computer-assisted research in the humanities, by contrast to the Cartesian story and traditional humanities practices, has almost always been collaborative. This is due to the variety of skills needed to implement digital humanities projects. It is also linked to the relationship between the practices of interpretation in the development of the tools of interpretation, be the tools for analyzing text or digital editions. Anyone who has used tools forged by another person is in collaboration, even if one isn't personally influencing the provider of the tools. The need to collaborate, though acknowledged in various ways, has been a professional hindrance, as anyone who submits a curriculum vitae for promotion listing nothing but co-authored papers knows.

    3. Pages 6-7

      Collaboration is not always good. It separates the interpreter/scholar from the designer/programmer who implements the scholarly methods. Willard McCarthy notes that the introduction of software "separated the conception of the problems (domain of the scholar) from the computational means of working them out (baliwick of the programmer) and so came at a significant cost.” As computing is introduced into research, it separates consumption, implementation, and interpretation in ways that can be overcome only through dialogue and collaboration across very different fields. Typically, humanities scholars know little about programming and software engineering, and programmers know little about humanities scholarship. Going it alone is an option only for the few who have time to master both. The rest of us and up depending on others.

    1. p. 8-actually this is link to p. 7, since 8 is excluded

      Another trend is the blurring of the distinction between primary sources, generally viewed as unprocessed or unanalysed data, and secondary sources that set data in context.

      Good point about how this is a new thing. On the next page she discusses how we are now collpasing the traditional distinction between primary and secondary sources.

  21. Jan 2016
    1. Is he saying something about inductive vs deductive methods? Where typically historians have a model or a hypothesis but now they are allowing the data to tell the story?

  22. Nov 2015
  23. Jun 2015
    1. The models criticized earlier do not need to be trashed. They are not just plain wrong. It's just that their sphere of applicability must be recognized as limited to a particular mode of exis­ tence, or a particular dimension of the real (the degree to which things coincide with their own arrest).

      This seems to be applicable to method discussions. Certain methods are applicable to certain audiences. Perhaps reductive...

    1. iterative-inductive research (that evolves in design through the study), drawing on a family of methods,

      Hmmm....I really like this articulation of method and find it analogous to "affect theory." That is...the slipperiness that we often encounter in affect theory is often a result of the formal commitments to an ongoing process of evolving terms. Here..."iterative-induction" works well to foreground that process of change.

  24. May 2015
    1. 'rbe concepts appear and reappellT like II revolving cast of characters, joining forces or interfer ing with each other in a tumble of abstract intrigues-at rimes (I admit) barely controlled

      I love the following few pages--on methodology?

    1. x = -a +/- (a2 -b)1/2

      This page looks very interesting but this kind of math formatting has forced many people to take their lives. $$x = -a \pm \sqrt{a^2 - b}$$

  25. Apr 2015
  26. scalar.usc.edu scalar.usc.edu
    1. get information of the literature’s abstr

      Do you mean that excluded literature published before the year 2000? This is also a methodological choice which effects results, thus it warrants some discussion of justification.

    2. vironment’,’trade’,’trad

      Why 30 times and not another cutoff? This kind of methodological choice makes a difference to results and thus deserves some discussion of its justification.

  27. Nov 2013
    1. In use these should be united, so that the same oration can expound purely, speak ornately, and express thought wisely. However, the precepts of pure diction, ornate delivery, and intelligent treatment must be kept separate and should not be confused.

      surely, like learning to play the guitar: fingering on the keyboard, strum and pick, and annotation are studied as distinct practices, and combined together to produce the music.

    2. method

      Method vs. purpose Grammar vs. message

  28. Oct 2013
    1. Moreover, we must not even trust to the first learning by heart; it will be better to have syllables repeated and to impress them long upon the memory; and in reading too, not to hurry on, in order to make it continuous or quick, until the clear and certain connection of the letters become familiar, without at least any necessity to stop for recollection

      Teaching to long memory, more effective than to short memory

  29. Sep 2013
    1. false knowledge

      substitutes "knowledge" for "learning"

    2. SOCRATES: And that, Gorgias, was what I was suspecting to be your notion; yet I would not have you wonder if by-and-by I am found repeating a seemingly plain question; for I ask not in order to confute you, but as I was saying that the argument may proceed consecutively, and that we may not get the habit of anticipating and suspecting the meaning of one another's words; I would have you develope your own views in your own way, whatever may be your hypothesis. GORGIAS: I think that you are quite right, Socrates.

      Socrates himself seems to be a master of persuasion via making the opinions of his opponents sound an awful lot like his own.

    3. Now I want to know about rhetoric in the same way;—is rhetoric the only art which brings persuasion, or do other arts have the same effect? I mean to say—Does he who teaches anything persuade men of that which he teaches or not?

      If this is that, then is that also this?

      Socrates method of persuasion seems to be to tease out distinguishing elements in such a manner as to expand the view and scope of the proposition. it feels like kind of a psychological exercise. I feel like he is going somewhere with this and that he has used several rhetorical tactics and tricks of persuasion that are about be revealed.

    4. And here let me assure you that I have your interest in view as well as my own

      not arguing for the sake of argument, but clearly in pursuit of truth - the psychology of his approach - his method