Diluent Buffers (A, B or C) are recommended for making dilutions of restriction endonucleases. When necessary, we recommend diluting enzymes just prior to use and suggest that the final concentration of diluted enzymes be at least 1,000 units/ml

Dilute the restriction enzyme using the recommendeddiluent buffer

DNA fragmentation by restriction digestion prior to dropletgeneration enables optimal accuracy by separating tandemgene copies, reducing sample viscosity, and improving templateaccessibility for input samples >66 ng per well

Digestion is recommended whenever DNA input is greater than 75 ng

The gun has been called the great equalizer, meaning that a small person witha gun is equal to a large person, but it is a great equalizer in another way,too. It insures that the people are the equal of their government wheneverthat government forgets that it is servant and not master of the governed

The sample can be periodically mixed by pipetting/vortexing/shaking to ensure thesbeadex particles remain in suspension. This mixing may increase the efficiency ofthe binding/washing

The composition of Buffer EB is: 10 mM Tris-Cl, pH 8.5

Size optimized Low settling rates for automation applications

low TE (<0.1 mM EDTA)

We recommend using a dsDNA quantification assay such as Qubit or PicoGreen. Note that the NanoDrop cannot distinguish between dsDNA and ssDNA (oligos and dNTPs) and may cause you to overestimate the amount of dsDNA in your samples.

Do not exceed 5,000 copies of target/μl of the final ddPCR reaction mix

Do not heat inactivate at greater than 65°C

Many assays will perform well regardless of which NEBuffer is used or the amountof 1x digest reaction loaded into ddPCR

Restriction Digestion

cell pellet suspended in 20 μl of Lyse and Go PCR Reagent (Thermo Scientific)

centrifuged at 10 000 g for 30 min to collect the cell pellets

Keep sorted transconjugants in 30% glycerol at 4C

3 mL pyrophosphate buffer

making our analyses available through a Shiny app and R package

Mix thoroughly and incubate for 5 minutes with periodic shaking

Google Photos ended its unlimited free storage policy for photos and videos as of June 1

Any Wanna Get Away or Wanna Get Away Plus funds will be forfeited

No change fees. We never charge a fee to change your flight

replaced the Taq polymerase used for primary amplification of the unique molecular identifier (UMI) tagged templates with a proofreading polymerase (Supplementary Fig. 16). This increased the percentage of error-free reads nearly four-fold, which greatly improves our ability to resolve full-length 16S rRNA ASVs (FL-ASVs) from low abundant taxa because at least two identical sequences are required to resolve an FL-ASV

1391r primer48 as it has better coverage of the known bacterial diversity

Dueholm, M. S. et al. Generation of comprehensive ecosystem-specific reference databases with species-level resolution by high-throughput full-length 16S rRNA gene sequencing and automated taxonomy assignment (AutoTax). mBio 11, e01557–20 (2020).

ASVs are not ideal as references for linking microbial identity with the physiology and ecology of key community members

India

if your treatments are ordered, don't compare each mean with each other mean (multiple comparisons), instead do one test for trend to ask if the outcome is linearly related with treatment number

e are typically not terribly concerned with Type 1 error because werarely believe that it is possible for the null hypothesis to be strictly true.

NNK

Supports up to 8GB maximum memory

new avenues for studying ARG transfer (Karimi et al., 2015; Li et al., 2018) (Figure 2C). Microfluidic chips equipped with delayed imaging, coupled with fluorescence technology, enable real-time observation of community changes and tracking of the transfer dynamics of ARGs

Scott Banta “Engineering of Acidithiobacillus ferrooxidans forBioelectrocatalysis Applications”

Akihiro Okamoto “Decoding Extracellular Electron Transfer: A NewGlimpse into In-VivoEnzyme Dynamics

Biki Kundu

10:20AM Biki Kundu

lmer(heartrate ~ depth + duration + (1|bird), data = penguins)

Use the lsb_release -a command to display the Ubuntu version.

quarto publish gh-pages document.qmd

A bracketed sequence of inlines, as one would use to begin a link, will be treated as a Span with attributes if it is followed immediately by attributes: [This is *some text*]{.class key="val"}

8.4.2 infer package workflow

A nice and easy way to report results of an ANOVA in R is with the report() function from the {report} package:

out-width Width of the plot in the output document, which can be different from its physical fig-width, i.e., plots can be scaled in the output document.

There are a number of ways to include dynamic variables within documents rendered by Quarto

I

This preparation includes fragments from the RNA-dependent RNA polymerase and VP1 (ORF1-ORF2 junction) regions.

(Cog1F and Cog1R for GI viruses; Cog2F and Cog2R for GII viruses) (27), and 200 nM each probe (Ring 1E, FAM-TGG ACA GGR GAY CGC-MGBNFQ, where FAM is 6-carboxyfluorescein and MGBNFQ is minor groove binder and nonfluorescent quencher

git log -SFoo -- path_containing_change

Callouts are an excellent way to draw extra attention to certain concepts, or to more clearly indicate that certain content is supplemental or applicable to only some scenarios.

This item type can also be adapted to fit many types of unusual items.

When a desired DNA is present at low concentrations relative to nearly identical undesired DNAs, LNAs can block amplification of unwanted DNAs

Ensuring that each amplicon is only copied by its “correct” SuperSelective primer

shorter foot lengths, though lowering the equilibrium abundance of foot hybrids, resulting in longer delays before the threshold cycle is achieved, lead to enhanced selectivity

Although single-color ddPCR assays have been already utilized to assess SNVs at low VAFs24, it is required to design two different allele-specific primers to detect one SNV (wild-type and mutation-specific assays)

SuperSelective primers, which enable the amplification of SNVs in the presence of an excess of the corresponding wild-type target

Underlined letters represent LNA bases.

All reference sequences (ABCC9 and SNV wt assays) were located in copy-number-neutral regions. Sequence details are available upon request.

SNP PI3Kca_E545K

An RMD assay comprises a single set of primers plus two competitive probes (each probe with a different fluorophore), one detecting the wild-type allele, and one detecting the variant allele.

Drop Off (DOF) Determines absolute quantification of targets for assays designed to detect non-wild type sequences, such as indels and genome edits. The experiment type isdesigned to support an assay strategy where one probe counts all alleles andone “drop-off” probe sits on top of a predicted cut site.

Tm enhancers for probes are recommended for single nucleotide polymorphism (SNP) and rare mutation detection assays in order to keep the background fluorescence to a minimum. Shorter probes discriminate better between single base differences in the target amplicon(s)

Relative expression level measured by RNA QASeq in four clinical FFPE tumor tissue samples and three normal placenta FFPE samples

10 ng Human PBMC gDNA from the same healthy donor was used per experiment, corresponding to 2,790 haploid copies

Windows 10 has a built-in calibration tool that you can use to fix various display issues associated with your monitor.

experiment with different screen resolutions within Windows 10 Settings until you get to one that looks the way you want.

You need to turn off overscan, either on the TV or in the graphics card settings.

With the file(s) selected, press Shift-Z

or changes between two files on disk

This approach exploits the equivalence between confidence intervals and hypothesis tests, detailed in Section 12.2.

we must add aes(y = ..density..)) to the call to geom_histogram, which rescales the histogram to have area 1 (just like a density function has). We can then add the density function using geom_function

The data on the 463 courses at UT Austin can be found in the evals data frame included in the moderndive package

a digestion with Plasmid-Safe DNase (Lucigen, Madison, WI, USA) was performed to purify the extracted plasmid DNA

Incremental reveal Not limited to just bullet points... Use `. . .` syntax to indicate a break at an arbitrary location . . . ```{r} head(mtcars) ```

To start and end a video, you need to use ?start=<seconds> and ?end=<seconds> to define the starting and ending times. Something like this:https://www.youtube.com/embed/xxx6x67ws7?start=45&end=200

10,000 units

Benefit from increased resistance to inhibitors and chemicals with our optimized buffer system

Any other educational assistance that is excludable from gross income (tax free),

Your filing status is married filing separately.

credits are based on the amount of adjusted qualified education expenses paid for the student in 2023 for academic periods beginning in 2023 or beginning in the first 3 months of 2024.

Qualified education expenses paid on behalf of the student by someone other than the student (such as a relative) are treated as paid by the student.

Scholarship - partially taxable. Amount spent on tuition and qualified education expenses (provide link) not taxable, the remainder is taxable ordinary income. Reported on 1098-T if from USA stipend is treated as a Scholarships

You cannot claim a credit for education expenses paid with tax-free funds. You must reduce the amount of expenses paid with tax-free grants, scholarships and fellowships and other tax-free education help.

a formal billing arrangement is an arrangement in which the institution: (1) bills only an employer or a governmental entity for education that the institution furnishes to a student

Asking questions

Midterm

git log --oneline --graph --decorate --all

git config --global alias.tree 'log --oneline --graph --decorate --all'

I use Bash shell .sh scripts in my Windows projects to automate Infrastructure as Code, then run these scripts using Windows Subsystem for Linux - WSL I need to make sure the line endings of my Bash scripts are in the Unix style LF or \n and not Windows CFLF or \r\n

. The gh-pages branch method is based on an old GitHub Pages publishing option. This branch is somewhat separate from the rest of the version control tree

250 μl of sample was loaded into the first well of each row in a 96-well plate, and 10-fold serial dilutions were made using a multichannel pipette (Rainin, Emeryville, CA) by transferring 20 μl from column i into 180 μl of medium in column {i+1}, mixing 10 times, and repeating the process; pipette tips were changed between dilutions

Decimal serial dilutions (100–1000 μl) of 101–106 or 107 were prepared from the 100 stock in 1.5 ml tubes with 4–5 repeated flushing and changing of tips

However, none of these approaches combines the simplicity, low cost, dynamic range and versatility of simply diluting cells and then growing drops on solid media as first proposed in ref. 17.

Pipet 100 μL of Starting Culture into Dilution 1. Discard tip. Do NOT pipette up and down. Vortex tube for 5-10 secs

The problem raised by the #barbarplot campaign is that bar plots are a poor summary of the distribution of data.

a kanamycin resistance gene known to be expressed in both Gram-negative and Gram-positive bacteria

Place a space before and after () when used with if, for, or while.

generally derives from variations in filtering out spurious and low-abundant sequences (e.g. Edgar, 2017; Prodan et al., 2020).

6c CAACGCGARGAACCTTACC 966–984

though 8 V/cm (120 V) seems to work fine for routine use

Since less alcohol is required for isopropanol precipitation, this is the preferred method for precipitating DNA from large volumes. In addition, isopropanol precipitation can be performed at room temperature, which minimizes co precipitation of salt that interferes with downstream applications

Summary of the experimental setup

Dashed line: Background fluorescence of cells before transduction.

the P2-STF chimera encoded on a plasmid (p938)

tail tip protein gpJ recognizes the LamB outer membrane porin and results in an irreversible binding of the phage to the cell surface

Efficient conjugative delivery to different strains and species will thus likely require different specialized systems

Full length Illumina tagged primers used in study:

using the FACS tosort for all three gates at highest speed by sorting for gain

What does a “model” mean?

why should researchers make websites? Because it is the easiest way to disseminate your work, your projects, who you are as a researcher, and to have more impact and outreach.

One of the most useful features of Quarto reveal.js slides is absolute position, which allows you to specifically place elements on a slide.

using the FastStart High Fidelity PCR System

515F GTGCCAGCMGCCGCGGTAA Universal Turner et al. 1999

PCR primers F-968-GC (59-CGC CCG GGG CGC GCC CCG GGCGGG GCG GGG GCA CGG GGG GAA CGC GAA GAA CCT TAC-39)

968f968–984AACGCGAAGAACCTTACNübel et al., 1996

In code, use comments to explain the “why” not the “what” or “how”.

Fluorescence Activated Cell Sorting (FACS).  From sorting cells for single cell genomics to identifying the host range of a plasmid; from detecting metabolically active cells in an environmental sample to studying colitogenic-driving intestinal bacteria, FACS can be used in a multitude of applications to study microbial communities (Rinke et al. 2014, Klumper et al. 2015, Kalyuzhnaya et al. 2008, Hatzenpichler et al. 2016, Palm et al. 2014).

zygotically inducible

we report on a new approach to quantify the fraction of a bacterial community that is able to receive and maintain an exogenous conjugal plasmid termed community permissiveness

This pool best represents the cells that had the potential to receive the plasmid, compared with the initial wastewater community before mating, i.e. before the few rounds of division that occur on the filter during incubation

Conjugation events were visualized by stereomicroscopy and quantified by automated image analysis

Filters were incubated for 48 h at 25 °C, before 72 h storage at 4 °C for GFP maturation.

a shows the sorting of the initial soil bacterial recipient community

(d) shows the enrichment of transconjugants after the first fast enrichment sorting step to over 80% transconjugal cells

To be right, it must be possible to be wrong

This model allows for the lessons to remain free from brand integrations and sponsor messages, without introducing paywalls.

I wash and dry my pan immediately after each use, but it’s what happens next that matters: I heat it gently over a low flame and add a bit of ghee. Then, I grab my dedicated “cast iron cloth” and rub the oil into the bottom and sides of the pan. I wait until the pan feels warm, but the ghee isn’t shimmering and snapping. Then I simply turn off the heat and let the pan cool naturally. This small step keeps it glossy and well-seasoned.

To save a copy of the slide with your drawings, your best option is to print your presentation from the browser.

When creating line art, please use the following guidelines:

White space must be cropped from the image, and excess space between panel labels and the image must be eliminated.

The blastn parameters used for VecScreen are significantly more stringent than the default blastn parameters

what economists call rents: that is, value extracted through the ownership of a limited resource

Any abuse of market power is likely to show up first on the supply side

The marketplace is designed and controlled by its owners, and that design shapes “who gets what and why” (to use the marvelous phrase from Alvin E. Roth, who received a Nobel prize in economics for his foundational work in the field of market design.

control the market through the algorithms and design features that decide which products users will see and be able to choose from. And these choices are not always in consumers’ best interests.

Likewise, Google does not just compete with other search engines like Bing and DuckDuckGo, but with everyone who produces content on the world wide web. Apple’s iPhone and Google’s Android don’t just compete with each other as smartphone platforms, but also with the app vendors who rely on smartphones to sell their products.

To do so, we prepared 96-well plates with two-fold dilutions of each antibiotic, ranging from 400–6.25 µg/ml, in 100 µl of medium per well

However, Google Docs has several limitations (like not automatically doing sequentially number headings - a plugin to do this available but is buggy) so I would like to do the final polishing in Word

Note: option -u was replaced by -b in Mash v1.1.

Download the pre-sketched RefSeq archive (reads not provided here; 10x-100x coverage of a single genome with anysequencing technology should work):refseq.genomes.k21.s1000.msh

P. lundensis is a Gram-negative, polar flagellated bacterium belonging to the Pseudomonas fragi clade of the Pseudomonas fluorescens species complex

sample size must be between 3 and 5,000 in order to use the shapiro.test() function.

RNA guides and corresponding target RNA, ranging from 25 to 373 nt, present signals

You can also create human-readable block identifiers by adding a blank space followed by the identifier, for example ^quote-of-the-day, at the end of a block:

30 - 60 sec/kb

When subjects are instructed togenerate a random sequence of hypotheticaltosses of a fair coin