...enzyme sites. You can easily move (subclone) any piece of DNA that already has restriction sites on either...systems is that like restriction enzyme sites, type IIS sites can be found throughout DNA sequences. Thus...double stranded DNA (dsDNA) at specific sequence sites in the DNA. In restriction cloning, scientists utilize...either side of it into any plasmid that has the same sites in the same orientation within its multiple cloning...short length, it is also easy to add restriction sites to any piece of DNA during PCR amplification, allowing...important to note that restriction enzyme target sites can be repeated throughout a specific DNA sequence...already be surrounded by specific recombination sites (in this regard, not so dissimilar from restriction...

... genuinely excites them, and reach out to the PI to get involved. It not only demonstrates initiative ...problems. Biomedical research, for instance, contributes heavily to medical practice, and understanding...tasks Principle investigator (PI) Runs the lab Writes a lot of grants Varying degrees of engagement with...helped me cultivate stronger relationships with my labmates. Sometimes what I thought was a dumb and very ...Google is a great resource too.) Connect with your labmates and mentors Many of the grad students and postdocs...resources for career advice and advancement, my labmates have been some of my closest friends and have ...often have scholarships and funds dedicated for undergraduates interested in doing research and attending ...

...ligate many undesirable structures, including substrates containing gaps or DNA base pair mismatches (...such as Taq DNA Ligase, which only reacts with substrates containing no gaps, and will not join any fragments...simultaneous interrogation of multiple potential SNP sites (15). The related LCR technique uses two sets of...generally prefer fully Watson-Crick base-paired dsDNA substrates to those containing one or more mismatches. However...pyrimidine mismatches are typically worse ligation substrates than pyrimidine:purine mismatches. Helix stability...naturally able to discriminate against ligating substrates containing base pair mismatches (i.e., are "higher...reduces the concentration of annealed mismatched substrates (expected to have a slightly lower Tm around ...

...your Eppendorf tubes, hang up your pipettes, and seal up those plates – it’s time to turn your attention...to announce the winners of the 2022 #DeckTheLab contest!  and it's..... Michael Früh submission, which...2022   Congratulations to all our #DeckTheLab contestants!           View this post on Instagram                     ...attention to decorating! To join the contest, you need only decorate your lab, take photos, and then share them... enter: Post your picture or video for either contest on Twitter or Instagram by December 31, 2022.  ...a lab supply tree and set a challenge to my lab mates. They did not disappoint. #deckthelab pic.twitter.com...

...dye-nitrilotriacetic acid (NTA) conjugates (Kapanidis, Ebright, & Ebright, 2001). These Cy/NTA conjugates work the same ... What are the proper controls needed to pressure-test your FRET assay to make sure it is working? This...fluorophores to the different biomolecules you’d like to test for interaction. In fact, targeting a FRET fluorophore...attached fluorophore. However, since tRNA suppression competes with natural chain termination at stop codons,... only in a few cell types (E. coli and Xenopus oocytes, mainly). Nevertheless, tRNA suppression plasmids...tag from 6 to 10 residues. While these Cy/NTA conjugates are not commercially available, they are easily... the donor and the acceptor fluorophore binding sites are adjacent to each other on the same protein and...

... of Antonello Bonci, Director of the National Institutes of Drug Abuse Intramural Research Program (NIH-NIDA... I work in), an obvious behavioral experiment to test for sufficiency of your circuit of interest in promoting..., on the other hand, would not be an appropriate test.  Of course, proper controls are required and include...sutures and subsequent determination of the proper coordinates of your brain region of interest (as shown in...identical for all of these surgeries. Identify coordinates through a rodent brain atlas or research papers...prior to using the virus. You may need to adjust coordinates based on your own method of identifying bregma...readout. The advantage is you can “zero” your coordinates at bregma which allows you to quickly and reliably...

...of DNA, or templates, are spatially separated from each other. These attached DNA templates are then amplified...Alternative start codons include CUG in eukaryotes and GUG in prokaryotes. Common Epitope Tags Tag Amino Acid...DNA. Although they can be found in archaea and eukaryotes, they play the most significant biological role...fragment with a plasmid or vector DNA backbone generates a recombinant DNA molecule, which can be used ...of DNA which contains several restriction enzyme sites, enabling easy insertion of DNA by restriction enzymes...the promoter. As a general rule, the restriction sites in the MCS are unique and not located elsewhere ...relatively quickly, and exhibit exponential growth rates, plasmids can be replicated easily and efficiently...

...regularly, and mix samples very well.  Use duplicates or triplicates to increase the accuracy of your experiment...viral vector production facility, food allergy testing lab, and the grad student down the hall from you...-fold dilution series. Each step of the series dilutes by 1:2 for a series that ranges from 1:2–1:32. ...standard dilution series in duplicate or triplicate. Replicates allows you to calculate the curve’s coefficient...replicate measurement divided by the mean of all the replicates multiplied by 100. To calculate the %CV of the...ideal %CV is less than 15%. You should also run replicates for all of the samples in the assay, as this ...these.  Review previous literature or results from labmates to get a sense of the concentrations you might...

...CNS cell types, including microglia, astrocytes, and oligodendrocytes, provided that reporter genes specific...activators to induce gene transcription at genomic sites targeted by specific sgRNAs. Its RNA-guided nature...packaging limits (i.e., how many sgRNA expression cassettes will fit). Cre-dependent expression of the SPH...selectively target these different transcriptional start sites in order to better understand isoform-specific gene...activity (hence, “immediate-early”) and consequently regulates neuronal physiology by promoting the development...gene possesses 9 distinct transcriptional start sites, all of which yield the same BDNF protein. To demonstrate...cell types (microglia are a notable exception) necessitates such molecular gymnastics for sensitive applications...

... bacterial ARG from Serratia sp. 39006. We next tested bARGSer with several inducible expression systems.../L yeast extract, and 5 g/L NaCl) or on LB-agar plates containing glucose and L-arabinose at 37oC for ... arabinose and colonies on arabinose-containing plates will be visibly white, and gas vesicles in cells...from left to right: colony whiteness on inducer plates, white inclusions in cells with GVs via phase contrast...mARGs also had a large genetic footprint (two cassettes + a booster cassette), their expression heterogeneity...mARGs come as a set of two PiggyBac integratable cassettes, both with doxycycline inducible promoters (Fig...increased ultrasound contrast. In all the cell lines we tested, 1 µg/mL doxycycline was sufficient to saturate...

... order fulfilment from an average of 40 minutes to 10 minutes. While users can’t tangibly see these improvements...each project. This creates accountability on the part of each developer and creates trust amongst the ...collaborate with the Product Team who in turn collaborates with stakeholders to resolve the questions.  ...would be staying on top of any third party library updates. Third party libraries are reusable software components...with their third party libraries and any security updates. Most of the time, updating these libraries is ...this year, which means there will be no further updates, security or otherwise. This might sound like Greek...

...called a Kirby-Bauer disk diffusion test or Kirby-Bauer susceptibility test (Figure 2), and requires at least...shows how to perform a Kirby-Bauer disk diffusion test and check bacteria for antibiotic resistance. Source...bacteria are causing the infection before they can test for resistance. Growing and isolating the infecting...infecting bacteria can take 24 hours or more, and testing for resistance often takes an additional 18-24 hours...available. For routine infections, like strep throat, testing for antibiotic resistance isn’t commonly performed...infections is so rare. When antibiotic resistance testing is requested clinically, this involves growing ... treated. We can prescribe antibiotics for the shortest time period possible to effectively manage a patient...

...restriction sites flanking the promoter-gRNA construct, but with different sequences adjacent to the sites. When...genome by removing the sequence between two target sites. Modifying multiple genes at once. Using multiplexed...promoter-gRNA constructs in order to target multiple sites! In this post, we'll cover Cas9 multiplexing options...oligonucleotides. These plasmids contain Type IIS restriction sites that flank the promoter-gRNA constructs. When these...unique overhangs (here, O1-4) adjacent to the cut sites “link” fragments together and drive ordered assembly...transcribed together and are flanked by specific sites that allow them to be cleaved and released. These...smaller than constructs with multiple promoter-gRNA cassettes, making them advantageous for small capacity vectors...

...personal litmus test was whether my wife could explain what I was proposing. The result of my test was humbling... your candidacy. A major reason that a lot of candidates either get no interviews, or get several interviews...accomplish. No matter how many times I say this, candidates regularly try to convince me that their search...language as possible. Seriously. My suggested litmus test is whether your friend in a lab outside of your ... and therapies." This is only the first few minutes of the chalk talk. Realize that it is rare to make...available here for free!) contains strategies, hints, anecdotes, and insights. After I wrote the first version... Jennifer Lippincott-Schwartz at the National Institutes of Health, was an Associate Professor at Albert...

...DNA with a TA dinucleotide (there are many such sites in the human genome). The original plasmid is empty... then degraded by the cell.4.) The transposase creates a double strand break in the DNA and allows the...has been designed to perform gene transfer in vertebrates. The system is comprised of 2 components: 1) ...number of extinct transposon sequences and then testing various combinations of changes. The basic functional.... The vectors each contain two SfiI restriction sites with different overhangs depending upon whether ... Feb 4. doi: 10.1002/biot.201400821. PubMed. 2. Mates L, et al. Molecular evolution of a novel hyperactive...transposase enables robust stable gene transfer in vertebrates. Nat Genet. 2009 Jun;41(6):753-61. Epub 2009 ...

... transcriptional units and facilitates integration. LEDGF/p75 binding sites are enriched in gene bodies...shuffling restriction sites between vectors until the gene of interest is flanked by sites compatible with ... that contain the restriction sites needed. Functional restriction sites must be a certain number of bases... always SIN: Uses 4 plasmids instead of 3 and eliminates the requirement for Tat. LTR Viral Promoter Wild...cells are usually used to produce lentivirus. What dictates lentiviral host cell range (tropism)? Lentiviral...target species. Although chromatin availability facilitates integration, it does not explain the lentiviral... subcloning or appending compatible restriction sites onto the insert of interest using PCR. The process...

... incubation (10–20 minutes) at 70 °C, or a truly lengthy incubation (30–60 minutes) at 37 °C, if you suspect...DNA-binding proteins, you may need to sonicate your lysates in order to release the proteins from their binding...in your background, try asking more experienced labmates or looking it up online. You’ll find many resources...your timer to ding. If you’re shaving off a few minutes here and there, and seeing high background in your...antibodies). You’ll want to run a protein gradient to test the antibody at the recommended dilution. If the...concentration decreases).     Figure 3: Testing the antibody’s dynamic range. A) The protein dilutions...from 1:100–1:1,000. If your antibodies come from ascites fluid, try a dilution range from 1:1,000–1:100,...

...growth and DNA yield. Test multiple, small colonies After streaking LB agar plates, you may notice that...vectors (LTRs and ITRs), plasmids with multiple polyA sites, and other plasmids that contain repeating elements...how to amplify recombination-prone plasmids and testing your prep can save you the headache of troubleshooting...cultures contain the recombined vector backbone. Test multiple colonies before saving a glycerol stock...you can try re-streaking for single colonies and testing multiple preps. In some cases, we suggest gel extraction...the recombined plasmid is still visible. This indicates that the DNA prep contains a mixture of the full...

...your incubation time on ice to 5 minutes (incubating the full 30 minutes will not significantly improve ...standard primers (no need to add unique restriction sites on the ends) and amplify your sequence of interest...the PCR product and TOPO Vector.  3. Incubate 5 Minutes at Room Temperature: You can place your reaction...digest, or sequencing. Pro tips Do not add 5’ phosphates to your PCR primers; you need that free hydroxyl...functionality are used in place of Taq to reduce error rates; however, proofreading polymerases will also remove...); however, you may need to incubate for 20-30 minutes if your PCR product is at a low concentration or...

...for the targeted gene. After initial testing, Dickinson et al. tested this system across 7 genes in parallel...remove after injection, they added flanking loxP sites and a heat-shock inducible Cre to generate a self-excising...worms have laid eggs, hygromycin is added to the plates to select for candidate knock-ins. These candidate... is inserted at the 5’ end of a gene, the SEC separates the targeted gene from its promoter, creating ...protein in the C. elegans genome. Although they tested fluorescent protein fusions, this system could ...editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans. Paix A, Wang Y, Smith...