127 Matching Annotations
  1. Jul 2019
  2. Jun 2019
    1. V8 protease-mediated semisynthesis of a globin was carried out at 4°C in 0.05 M ammonium acetate buffer (pH 6) containing 30% 1-propanol. For this, the lyophilized samples of natural or synthetic analogs of a 1-30 and respective a31-141 were individually prepared in water. Suitable volumes of the complementary fragments were mixed to obtain a 1:1 molar ratio and lyophilized. The lyophilized material was dissolved in appropriate amount of ammonium acetate buffer (pH 6). To this solution, a suitable volume of 1-propanol was added to a final concentration of 30% 1-propanol and 20 mg/ml substrate. The mixture was cooled on ice subsequent to which suitable volume of V8 protease solution prepared in water ( 1% w/w of substrate) was added. The ligation reaction mixture was incubated at 4°C for 24 hours. The extent of synthesis was monitored on RPHPLC by loading an aliquot of the reaction mixture on an analytical reverse phase column. The reaction was stopped by addition of chilled 5% TF A solution (0.2 fold v/v) and lyophilized
    2. Construction of mutant a globins
  3. May 2019
    1. DTT. Then contents were then mixed and 1μl (200 units) of M-MLV was added. The mixture was then incubated at 37°C for 50 minutes. The reaction was stopped by incubating the mixture at 70°C for 15 minutes. The cDNA thus prepared was then used as a template for PCR. The expression of the investigated genes was determined by normalizing their expression against the expression of actin or GAPDH gene
    2. Semi-quantitative RT-PCR
    3. μg of total RNA was reverse-transcribed using poly-T oligonucleotide and M-MLV reverse transcriptase (Invitrogen) according to manufacturer’s protocol. Briefly, a 20μl reaction volume was made for 1μg of RNA. In a microcentrifuge tube, 1μl oligo (dT)(500μg/ml) , 1μg total RNA, 1μl 10mM dNTP mix and sterile water was added to afinal volume of 13μl. The mixture was then incubated at 65°C for 5 minutes ad quickly chilled on ice. To this mixture were added 4μl of 5X first strand buffer and 2μl of 0.1M


    1. Activation of H50 protein chip array
    2. b. 5 μl of ACN + TFA (25% ACN in PBS + 0.1% TFA) was added to the spot surface and removed after 30 sec. c. 5 μl of cell lysate sample was then spotted on the chip and kept in a humid chamber for 30 min. d. Stringent washes were given by spotting 5 μl water on the spot surface for 30 sec and removing using Whatman filter paper strips. This was followed with a 25% ACN wash or three washes with 25% ACN or 50% CAN or 75% ACN. e. Washing was performed by spotting 5 μl of water for 30 sec followed by removal using Whatman filter paper strips. f. Dried chip at room temperature. g. 1-2 μl of SAP matrix (5 mg of matrix + 200 μl ACN + 200 μl of 1% TFA) was then spotted on the chip surface and allowed to dry. h. The chip was then placed in the SELDI machine
    3. 5 μl of water was added to each spot on the chip and removed after 30 sec using Whatman filter paper strips. Care was taken not to touch the spot surface. This step was repeated once
    4. Typically, 100-200 ng of DNA was used in each ligation reaction. The ratio of vector to insert was maintained between 1:3 and 1:5. The reactions were usually done in a 10 μl volume containing ligation buffer (provided by the manufacturer) and 0.05 Weiss units of T4 DNA ligase, at 16°C for 12-16 hr
    5. Ligation of DNA
    1. Primer extension analysis to map thetranscription start site was carried out as describedby Conway et al. (1987) and Rajkumari et al. (1997). 20 pmolof primer was labelled at its 5′-end with 32P-γ-ATP as described above. 106cpm equivalent of labelled primer was mixed with 10μg of total cellular RNA. Sodium acetate pH-5.5 was added to a final concentration of0.3 M and the nucleic acids were precipitated with ethanol, washed with 70% alcohol,air-dried and dissolved in hybridization buffer (9 mM Tris-Cl, pH-8 and 0.35 mMEDTA) and incubated overnight at 43ºC for annealing. Reverse transcriptase reactionwas performed by the addition of 5 mM MgCl2, 1 mMdNTP’s, 1 X RT buffer, highconcentration (10 units) of Superscript III Reverse Transcriptase (Invitrogen) to the mixture of annealedlabelled primer and RNA. The reaction was incubated at 43ºC for 1-hr following whichthe nucleic acids were precipitated with absolute alcohol and 0.3 M CH3COONa, pH-5.5. The precipitate was air dried and dissolved in water and gel-loading dye (95%formamide, 20 mM EDTA, 0.05% each of xylene cyanol and bromophenol blue) wasadded. The samples were heated at 90ºC for 2-min before loading on a 6% denaturingpolyacrylamide gel for electrophoreticresolution alongside a sequencingladder
    2. Primer extension analysis
    3. PCR products were purified using the PCR Purification Kit (Qiagen) as per the manufacturer's instructions
    4. Purification of PCR products
    5. A differential gene expression microarray with respect to argP was performed by Genotypic Technology Pvt.Ltd., Bengaluru. The experiment was performed on an oligonucleotide microarray having 10828 probes for coding region(on average three probes were designed for each 4294 coding regions) and 4380 probes for non-coding region (on average two probes were designed for 2240 non-coding regions). The RNA was labelled using Cy3 and single channel detection was used. Data was analysed using GeneSpring GX Version 7.3
    6. Microarray details
    1. Intracellular reactive oxygen species (ROS)levels in yeast cells weredetermined usingfluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA; Sigma# D6883). Cellular esterasesremove the diacetate groups ofthe DCFH-DAand produceDCFHwhich getsreadily oxidized to highly fluorescent product 2′,7′-dichlorofluorescein (DCF) by intracellular ROS. The fluorescent intensity of DCF corresponds to the amount ofintracellular ROSpresent in the cell.Cells grown under different environmental conditions were harvested,washed once with tissue-culture grade phosphate-buffered saline (PBS) and resuspendedin PBS to the final cell density of 1 OD. Freshly-prepared DCFH-DA (0.01M stock in DMSO) was added to the cell suspension toafinal concentration of 100 μM. Cell suspension was mixed and incubated at 30 ̊C for 30 min. After incubation,cells were washed 2-3 times with 1 ml PBS and then resuspendedin 200 μlPBS. Fluorescence intensity values wererecorded usingspectrofluorophotometer (Varioskan flash-3001, Thermo Scientific) with excitation and emission at 488and 530 nm,respectively.Fluorescenceintensityvalues obtained from probe-loaded cells were subtracted from the fluorescence intensity values obtainedfrom cells-alone samplesto remove background fluorescence
    2. Determination of intracellular reactive oxygen species (ROS)levels
    3. C. glabrataTn7insertion mutantlibrary was screened for reduced growth in YNB-pH 2.0 medium. Thismutant library,composed of 9,134 Tn7insertion mutants, isarrayed in 96-well microtitre plates(Castaño et al.,2003). 2 μlof each mutant strain was inoculated in 120μl YNB medium and grown overnight at 30 ̊C in an incubator with constant shakingat 120 rpm. Overnight grown cultures were 120-folddiluted with 1X PBS in a 96 well block and transferred, using a 96-well pin replicator, to YNB and YNB-pH 2.0 medium. Plates were incubated at 30°C and mutant phenotypes were recorded after 3-4days.
    4. Screening of C. glabrataTn7insertion mutants
    5. suspension was kept on ice for 10 min and 50 μl volume was aliquoted to chilled sterile microcentrifuge tubes. Cellswere immediately snap-frozen in liquid nitrogen and stored at -80 ̊C
    6. A single colony of E.coli DH5-α strain was inoculated in 10ml LB medium and incubated at37 ̊C for overnight. 4 ml of thisovernight culture was inoculated in 2 lt SOB medium and incubated at 18 ̊C till theOD600reaches to 0.5. Cells were harvested by centrifugation at 2,500 g for 10 min at 4 ̊C and washed gently in 80 ml ice-cold Inoue transformation buffer. Cells were collectedby centrifugation at 2,500 g for 10 min at 4 ̊C and gently resuspended in 20 mlice-cold Inoue transformation buffer. To this cell suspension, 1.5 ml sterile DMSO was added and swirled gently. Cell
    7. E. coli DH5α ultra-competent cells preparation
  4. sg.inflibnet.ac.in sg.inflibnet.ac.in
    1. Colony blot assay was performed to analyse secretion of carboxypeptidase Y(CPY)as described previously (Roberts et al., 1991). Single colony of a C. glabratastrain was inoculated in YPD medium andculture was grown till stationary phase. 0.1 OD600equivalent cellsfrom this culture were spotted on CAA medium,overlaidwith a nitrocellulose membrane and plate was incubated at30 ̊C for 18-20 h.Afterincubation, nitrocellulose membranewas washed with water to remove cells and membrane-bound CPYwas detected by immunoblotting with polyclonal anti-CPY antibody at a dilution of 1:10,000
    2. Colonyblot assay
    3. Stripping of membranes in buffer containing 0.4 M NaCl yielded slightly better results. Hybond membranes were reused for 5-10 times after stripping
    4. Radiolabeled-bound probes were stripped from the membrane by boiling in 1% SDS containing 0.1X SSC for 15 min. Alternatively, membraneswereincubatedtwicein stripping solution (0.4 M NaOH)at 45°C for 30 minto remove the bound probes
    5. Stripping of probes from hybridized membranes
    6. 24 h post infection, THP-1 macrophages were washed thrice with PBS, lysed in water and recovered yeast cells were used to infect THP-1 cells at a MOIof 1:10. Three rounds of macrophage infection foreach mutant pool were carried out to enrich for the desired mutants in the final population. The lysate of 3rdround infection was inoculated in YPD medium for overnight (output). Cells were harvested, genomic DNA isolated from each input and output cell pellet andunique signature tags were PCR-amplified with P32-labeledα-dCTP using primers complementary to theinvariant region flanking each unique tag sequence. LabeledPCR products were denatured at 95°C for 10 min, chilled on ice and were hybridized tonylon membranescarrying immobilized plasmid DNA containing 96 unique tagsfor 14-16 h at 42°C.Membranes were washed twicewith 0.1X SSC bufferand exposed to phosphorimager screen for 2-4 h. Radioactive counts for each spot were quantified using Image Quant and Fuji Multi Gauge V3.0 software. Relative percentage intensity for individual spot was calculated with respect to allspots present oneach hybridizedmembrane
    7. YPD-grown cultures (0.05 OD600) of each mutant pool (96 mutants, each carrying a unique signature tag) were either inoculated in YPD medium for overnight (input) or used to infect differentiated THP-1 cells (1X106). After 2 h incubation, non-cell-associated yeastcellswere removed by washing THP-1 cellsthricewith PBS. At
    8. Screening of C. glabrataTn7insertion mutant library
    9. THP-1 cells were seeded ina 24-well tissue culture plate to a celldensity of 1 million cells per well,treated with PMA and were infected with yeast cells to a MOIof 10:1. Two hours post infection, cells were washed thrice with PBS and medium was replacedwith fresh prewarmed RPMI medium.Plates wereincubatedat 37ºCfor 24 h. Supernatants were collected,centrifuged at 3,000 rpm for 5 minto get rid of particulate matter,if any, andwerestored at -20°C until use. Estimation of different cytokines wasperformed using BD OptEA ELISA kits as per the supplier’s instructions
    10. Cytokines measurement
    1. competent cells pre-inoculum was prepared. A single bacterial colony was picked from LB agar plate that has been incubated for 16-20 hours at 37 °C and inoculated into 3 mlLB medium and incubated overnight at 37 °C temperature with 200 rpm shaking. 1% of this pre-inoculum was sub cultured in 100 ml LB-broth and incubated at 18 °C until OD 600 reached 0.5 -0.6 (approx.). Culture was kept on ice for 10 min. with constant shaking. Cells were pelleted by centrifugation at 2000xg/4°C/8 min. Pellet was resuspended in 40 ml of ice-cold Innoue buffer. Bacterial suspension was kept on ice for 30 min, re-spun at 2000 xg/4°C/8 min. Pellet was resuspended in 8 ml of TB buffer inwhich final concentration of DMSO was 7% and left on ice for 10 min. 100μl aliquots were made and snap frozenin liquid nitrogen and stored at -80 °C
    2. All the salts (10 mM PIPES, 15 mM CaCl2.2H2O, 250 mM KCl,55 mM MnCl2. 2H2O) except MnCl2were dissolved in water and pH was adjusted to 6.7 with 1N KOH. MnCl2was dissolved separately in water. MnCl2was added drop wise while stirring (MnCl2if added directly will give a brown colour to the solution and precipitates;hence it needs to be dissolved separately). Solution was then sterilized by filteringand stored. To prepare
    3. PreparationofUltra competent cells
    4. Equal amount of proteins were loadedon an appropriate percentageof denaturing SDS-PAGE gel. After completion ofthe run, the gel was over laid on a PVDF membranecut to the size of gel and sandwiched between filter paper sheets and kept inthe blotting cassette in the presence of transferbuffer. Finally the cassette was put in themini transblotapparatus and blotting was done for 2-3hours at a constantvoltage of 80Vat 4⁰C. For blocking the nonspecific sitesmembrane was incubated with blocking solution(5% non-fat milk solution in TBST)with gentle shaking for 1 hourat room temperature. Excess milk from the membrane was washedoff with TBST and themembrane was incubatedwith primary antibody diluted in 1XTBST for 3 hours atroom temperature or overnight at 4°C withshaking. After incubation the membrane was washedwith TBST and incubatedwithappropriate secondary antibody (conjugated with horse-radish peroxidase)diluted in5% fat free milk solution (in TBST) for 1hat room temperature.The blotwas later washed thricefor 10min eachwith TBST and processed for the detection of proteinsignal using ECL-prime chemiluminescencedetection reagent followed by detectionof signal either on X-ray filmor in a chemiluminescence detectionsystem(Proteinsimple, California, USA)
    5. Immunoblotting
    1. Materials and Methods462.2.5 Cell proliferation assayThe method described earlier by Gilliesand co-workerswas slightly modified and followed (Gillies et al.,1986). Briefly, parentaland profilin-stable cells were seeded in triplicates at a density of 20,000 cells per well of a 24-well culture plates. Each day after seeding, cells were washed with PBS and stained with 0.2% crystal violet in 2% ethanol for 15 minutes. Vigorous washing was done with PBS to remove excess dye. Crystal violet dye was then eluted using 1% SDS solution with extensive pipetting and diluted 10 fold. Absorbance of the extracted dye was then determined at 570 nm in a spectrophotometer. Absorbance data based on triplicate set of samples for each experimental condition were then averaged for each time point to generate a growth curve.2.2.6 Preparation of whole cell, cytoplasmic and nuclear lysatesIn order to extract the total cell homogenate, the culture media was removed and cells were washed with ice cold PBS. The cells were then gently scrapped and pelleted by centrifugation at 3000 rpm for 3 minutes at 4°C. The cell extraction buffer was added to the cell pellet and placed on rotor kept in cold room for 30 minutes for cell lysis. Lysed cellswere then centrifuged at 13000 rpm for 10min at 4°C. The supernatant was collected as cellular lysate. Protein concentration was estimated using Bradfordassay, described below.For a typical Western blot assay, 30-70μg of protein was loaded on theSDS-PAGE.For the preparation of cytoplasmic lysate,ice-cold hypotonic cytoplasmic extract buffer was added in the cell pellet andgently mixed with the pipette in a microfuge tube. The cell suspension was incubated on ice for 30 minto allow them to swell.After incubation, freshly prepared 10% NP-40was added andvortexed vigorously for 15 seconds torupture the plasma membrane. The contents were then centrifuged at 13000 rpmfor a minuteat 4°C and supernatant containing the cytoplasmic lysate was transferred to another pre-chilled microfuge tube and stored at –70°C.The pellet was then further processed for extraction of nuclear lysate.For this, ice-cold nuclear extractbuffer was added to the pellet and incubated on ice for 45 min with intermittent vortexingafter every 10 min of incubation. Finally, cell suspension wascentrifuged for 5 min at 14000rpm. The supernatant containing nuclear lysatewas stored at –70°C for further experiment
    2. Preparation of whole cell, cytoplasmic and nuclear lysates
    1. Yeast weregrown in YPD (Difco) overnight,and sub-cultured at 0.2 OD600. Cells were harvested at 0.6-0.8 OD600. 1 OD600of each culture was used for the labelling. Cells were washed in SC-Metmedium twice, suspended in SC-Metmedium containing 25μCi/mLof 35S Met-Cys and pulsed for 15 min. Cells were washed twice in methionine-free medium and suspended in 300Lof Tris-saline. Cell suspension was counted in a liquid scintillation counter (Perkin Elmer-Tricarb 2900). The cpm values obtained were plotted using GraphPad Prism5
    2. 35S-Met uptake assay
    1. Plasmids containing the shRNA of interestwere either transfected transiently or were stably transfected. Transient transfection of shRNA was performed using eitherLipofectamine 2000 or PEI (as per the method explained before). Stable integration of shRNA was performed by transfecting shRNA along with retroviral packaging vector PCL-Ampho into BOSC23 packaging cells. The supernatantcontaining the packed viruses (viral medium)was collected at 48 and 72 hours of transfection. The viral mediumwas then added to thetarget cells in the presence of polybrene (8μg/mL). Two days later, cells were cultured in medium containing puromycin for the selection of stable clones.The clones stably expressing the desiredshRNA were identifiedandverified through western blotting and immunostaining using specificantibodies. A similar protocol was used to generate stable cell lines that expressed control shRNA
    2. ShRNA
    3. Cells were plated in a manner that they were 30-50% confluent on the day of transfection.Cells were washed with serum-free medium,and the serum-free medium was added to the cells as per plate size. SiRNA was diluted in the serum-free medium, and oligofectamine was diluted in serum-free media, separately (Table 10). Both the complexes were incubated at room temperature for 5 min. Diluted siRNA wasmixed gently with diluted oligofectamine and incubated at room temperature for 15 min. The final transfection mixture was added dropwise to the cells and mixed properly by gentle rocking. Cells were incubated for 4 hrs.,and the growth medium containing 10% FBS was added to the plates without removing the previous medium. Cells were incubated overnight at 37°C in a CO2 incubator. After overnight incubation, the siRNA transfection was repeated using the same protocol. Cells were harvested after 24-48 hours of second round siRNA transfection. The knockdown was detected bychecking the protein levels throughwestern blotting. (Note: SiRNA transfection is carried out in antibiotic free medium)Table 10: SiRNA transfection methodology
    4. SiRNA
    5. RNA interference
    1. In planta growth assay for different strains of Xanthomonas oryzaepv. oryzicolawas performed by counting CFUs. For getting the CFUs, 1 cm2 leaf area surrounding the site of inoculation was cut and surface sterilized by dipping the leaf in 1% (vol/vol) sodium hypochlorite for 2 min followed by three washes with sterile water. To get the CFUs, sterilized leaves were crushed using mortar and pestle, and diluted appropriately for plating on PSA plate containing suitable antibiotics for differentially marked strains
    2. In plantabacterial growth assay
    3. strain or the ∆rpfFmutantharboring the Wild-typeallele in plasmid (pSC9).Genes that were significantly up regulated by 0.6 or more or down regulated by -0.6 or less fold (log2–fold change) were identified.The microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) under the GEO series accession number GSE53255
    4. 8x15k (AMADID: 25096) custom Agilent platform comprised of coding sequences for the three strains of Xanthomonas-X. oryzaepv. oryzae(KACC10331), X. oryzaepv. oryzicola(BLS256) and X. axonopodispv. citri 306 gathered from National Center for Biotechnology Information (NCBI). A total of 8113 probes were designed wherein 2120 probes corresponding to genes of interest replicated three times on Agilent platform. Feature extraction software GeneSpring GX version 10.5.1 of Agilent and GeneSpring GX percentile shift normalization was used for data analysis. Genes that were significantly up or down regulated by more than 1.5 fold and less than 0.5 fold were identified. Hierarchical clustering was performed for the differentially regulated genes and classified based on functional category. Data are the average of two hybridizations from biological replicates of each sample andraw data sets for this study are available at the Gene Expression Omnibus database (Accession number –GSE217809). Likewise, Microarray analysis for Xanthomonas oryzaepv. oryzicolawas performed by isolating RNA from the strains grown under low-ironcondition. The labeled cRNA samples were hybridized on to a Genotypic Technology Private Limited designed 8x15k (AMADID: 41087) Agilent platform. Data extraction from Images was done using Feature Extraction software v 10.7 of Agilent. Data normalization was done in GeneSpring GX using 75thpercentile shift and normalization to specific samples. Differentially regulated genes were clustered hierarchically to identify significant gene expression patterns.Genes were classified based on functional category. Hierarchical clustering of DSF regulated genes in X. oryzaepv. oryzicola grown under low-iron conditions is based on similar expression profiles in ∆rpfFmutant vs either the Wild-typeBXOR1 strain or ∆rpfF(pSC9). Clustering analysis was performed using GeneSpring GX Software using Average Linkage rule with pearson uncentered distance metric. log2–fold change differences between the ∆rpfFmutant with either the Wild-typeBXOR1
    5. Xanthomonas oryzae pv. oryzae strains grown in PS medium to an OD600of 1, were collected, washed once with 150 mM sodium chloride (NaCl) solution to remove excess EPS. RNA isolation was performed using Trizol method described above. After isopropanol precipitation, RNA was frozen at -80°C. Quality of RNA was examined by determining the RNA integrity number (RIN) before microarray analysis. Microarray experiments were performed at Genotypic Technology Pvt. Ltd., Bangalore.Briefly, a
    6. Microarray analysis
    7. shaking at 200 rpm. 1% of overnight grown culture was inoculated in 100 ml fresh PS medium and grown to obtain log-phase culture. Log phase Xanthomonas culture was kept on ice for 10-15 min, aliquoted in 50 ml pre-chilled centrifuge tubes and centrifuged at 4000-5000 g at 4°C for 10 min. Supernatant was discarded and pellet from each tube was gently resuspended in 10-20 ml sterile chilled water. Next, cells were harvested by centrifugation at 4000 g at 4°C for 10 min and supernatant was discarded. Harvested cells were washed twice and finally resuspended in adequate amount of prechilled sterile water. 100 μl of cell suspension was aliquoted in sterile 1.5 ml microcentrifuge tubes and kept on ice. For transformation, Xanthomonaselectrocompetent cells and appropriate amount of plasmid DNA was mixed, and kept on ice in laminar hood. This mixture was added to 1 mm electroporation cuvettes (Biorad) and tapped gently to allow the cells to settle properly in order to avoid air bubbles. Competent cells were electroporated (1800 V, 25 μF, 200 Ω, 1mm cuvette) followed by immediate addition of fresh PS broth in the cuvette, mixed properly and taken in the microcentrifuge tubes. Microcentrifuge tubes containing transformed cells were incubated at 28°C for 2 hours with continuous shaking for recovery. After recovery, cells were plated on specific medium with appropriate antibiotics and incubated in 28°C plate incubator
    8. For electrocompetent cell preparation, single colony of desired Xanthomonasstrain was inoculated in 5 ml PS medium and grown overnight at 28°C
    9. Xanthomonastransformation
    1. Overnight-grown C. glabratacells were freshly inoculated either in YNBmedium or YNBmedium supplemented with BPS (50 μM) or FeCl3(500 μM) and allowed to grow for 4 h at 30°C, 200 rpm. After 4 h growth, cells were spun down at 4,000 rpm for 5 min in a refrigeratedcentrifuge set at 4°C and total protein was isolated. For estimation of histone deacetylase (HDAC) activity, 40 μg of protein samples were taken and HDAC Fluorometric Activity Assay Kit (#10011563; Cayman Chemical Company, Ann Arbor, MI, USA) was used as per manufacturer’s instructions. Fluorescence intensity values obtained inthepresence of the HDAC inhibitor, trichostatin A, were subtracted from those of the samples without inhibitorand plotted as relative arbitrary fluorescence units
    2. Estimation of histone deacetylase (HDAC) activity
    3. Bacterial plasmid DNA was isolatedusing the QIAprep® spin Miniprep kit (QIAGEN, 27106). 10 ml of LB medium supplemented with appropriate antibioticswas inoculated withasingle bacterial colony and incubated at 37°C for 12-16 h. Cultures were spun down at 8,000 rpm for 5 min, supernatant was discarded and the cell pellet was processed to isolate plasmid DNA as per instructions given in the kit. DNA was eluted either in nuclease-free water or in elution buffer provided in the kit and stored at -20°C until use
    4. Plasmid isolation
    5. Bacterial transformation
    6. Bacterial plasmid DNA was isolatedusing the QIAprep® spin Miniprep kit (QIAGEN, 27106). 10 ml of LB medium supplemented with appropriate antibioticswas inoculated withasingle bacterial colony and incubated at 37°C for 12-16 h. Cultures were spun down at 8,000 rpm for 5 min, supernatant was discarded and the cell pellet was processed to isolate plasmid DNA as per instructions given in the kit. DNA was eluted either in nuclease-free water or in elution buffer provided in the kit and stored at -20°C until use
    7. Plasmid isolation
    8. E. coliDH5α ultra-competent cells were used for all bacterial transformations. Briefly, frozen DH5α ultra-competent cells were taken out from -80°C freezerandthawed on ice for 15 min. DNA to be transformed was added to the bacterial cell suspension and incubated on ice for 30 min. For transforming ligation mixtures and plasmids,5-10 μl and 100-500 ng of DNA was used, respectively. Followed by 30 min incubation on ice, heat shock was given for 60-90 sec at 42°C in a water bath and cells were immediately kept back onice for 2 min. To this,1 ml of sterile LB medium was added and tubes were incubated inashaker incubator set at 37°C, 200 rpm for 45 min.Next, cells were spun down and resuspended in 500 μl of LB medium. About 100-200 μl of resuspended cells were plated on LB-agar medium containing appropriate antibiotics and incubated for 12-16 h at 37°C. Transformants were purified on LB-agar plates containing appropriate antibiotics andpositive transformantscarrying desired DNAwere verified by PCR, restriction digestion and sequencing analyses
    9. Bacterial transformation
    1. ncubated with appropriate Alexa-488 conjugated secondary antibodies diluted in PBST with 2% BSA at room temperature for 1 h (for dilutions refer to Table-2.3). The cover slips were washed with PBST, mounted on glass slides using Vectashield mounting medium with DAPI. Images were acquired on a LSM 510 META confocal microscope (Zeiss, LSM acquisition software, 63x 1.4 N.A. objective) at a 0.7 scan zoom to collect maximum number of cells per field. The number of foci in each nucleus was manually counted by changing contrast identically across all the samples. A minimum of 10 random fields were imaged per sample. Data are represented as average number of foci per nuclei.
    2. Localization of the DNA damage response proteins γH2AX, Rad51 and BLM and mitotic marker H3S10 (Histone H3 phosphorylated on Ser10) upon genotoxic stress and recovery was analyzed by immunofluorescence, following hypotonic lysis. Cells were seeded on cover slips in 12 well plates at 10-15% confluenceand incubated overnight. Cells were treated with 0.5 mM hydroxyurea and 0.25 μg/mL neocarzinostatin for 12 h. Coverslips were washed twice with PBS and cells were incubated in hypotonic lysis buffer containing 10mMTrisHCl (pH 7.4), 2.5mM MgCl2, 1mM PMSF and 0.5% NP-40, on ice for exactly 8 min on a shaker at 50-60 rpm. Cells were washed twice on ice, with ice cold PBS for2 min, with continuous shaking. Fixation was carried out with ice-cold 100% ethanol (which hadbeenkept overnight in -20°C) for 4 min, shaking on ice. 500 μLof ethanol was added to each cover slip during fixation. Cells were washedthrice with 1 mL PBS containing 0.2% Tween-20 (PBST), for 8 min each,at room temperature with shaking.Nonspecific interactions were blocked by incubating the cells for 30 minwith 2% BSA diluted in 1X PBS. Cover slips were again washed twice with 1 mLPBST, incubated with appropriate primary antibody (Table-2.3) diluted in PBS + 0.2% BSA for 2 h at room temperature. 200 μLof antibodywasplaced directlyonthecover slips. Post incubation, the cover slips were washed thrice with 1 mL PBSTfor 3 min each and the cells were
    3. Immunofluorescence