...necessary.   Tips for barcoding and tracking samples 1. Start with the tube - Barcoded tubes come in many...packages have spent days to weeks in transit and some have been transported across oceans and continents. Each... treated to easily take in plasmids (transformation). Transformed bacteria are then grown up on antibiotics...received is immediately transformed and stored. Some samples need to be transformed with a micropulser using... performs an average of 140 transformations. The lab typically transforms newly deposited samples from...delivered by a mail courier and then the journey of transformation and storage begins. Some samples are submitted...long-term storage in our facilities, every sample is transferred to a 2D barcoded tube (see next section) and ...

... such a popular model organism? Zebrafish (Figure 1) were initially recognized as a useful vertebrate ...strategy uses a transposon called Tol2 to randomly integrate foreign DNA into the genome. Non-random integration...et al., 2019). Generating zebrafish transgenics Creating transgenic organisms by introducing foreign DNA...Danio rerio) have been used since the 1930’s in a range of biological studies, including investigations ... to produce abundant, externally fertilized, transparent progeny (Figure 2). These features give it a ...individual to maintain. Also in contrast to mice, the transparent zebrafish embryo develops outside of the mother...sequenced genome also permits “whole-genome/transcriptome” studies, in which, for example, the activity...

...impacts of neural circuits on behavior. Figure 1: An overview of the chemogenetics system. Why use...approved drugs so that your results are viable for translation to human patients. Picking receptors The receptor... specifically designed Gi-coupled receptor in transgenic mice. Nature Biotechnology 17:165–169 . https...

...inclusion bodies and is folding properly. References 1. Angov, Evelina, et al. "Heterologous protein expression...structures in RNA transcripts Effects on transcription (remember, it’s not all about translation - e.g. codon... you cannot choose among the possible codons at random and expect your sequence to express well in any...therefore may need fewer resources for the entire translation process. For example, during high growth rate... tRNAs. For example Tuller et al. found that translation efficiency is well correlated with codon bias...tRNAs, different regions of the protein may be translated at different rates. The ribosome will move quickly...the ribosome stalls, this may give the swiftly translated regions a chance to fold properly. For example...

... education and scientific writing.     References 1. Martz, Colin A., et al. "Systematic identification...’s group at the University of California, San Francisco, is approaching the Ras problem by exploiting ...lines. Further, for each plasmid in the kit, the transcript most commonly found across all TCGA tumor samples...

...plasmids are passed down through generations (Figure 1). These so called “plasmid addiction systems” require...required for hok translation. Here, Mok and Hok translation is coupled. Translation from the Shine-Dalgarno...more stable than the methyltransferase. When a plasmid is lost, the methyltransferase is degraded and/or...systems have been used as biological tools for a wide range of applications in molecular biology or in vivo ...ccdB gene die off and a higher percentage of transformants contain the ccdB-less construct. Other toxin-antitoxin...composed of restriction endonucleases and methyltransferases. Restriction endonucleases cleave DNA at ...specific nucleotide sequences. To counter this, methyltransferases add methyl groups to bases at specific nucleotide...

...such as model systems and primary cells. References 1. Doench, John G., et al. "Rational design of highly...prevent efficient transcription from sgRNA targets. CRISPRi is effective in a narrow range around the transcription...two sets of sgRNAs; A, the top 3 ranked sgRNAs, and B, the next 3 ranked sgRNAs. Dolcetto was able to discriminate...Cas9 derivatives. Unmodified Cas9 causes double stranded DNA breaks that commonly result in a complete ... lab selected sgRNAs in this optimal window and ranked them based on their optimized sgRNA rules and potential...gene expression, dCas9 can also be used for transcriptional activation. Activation of specific genes allows... strategies have been developed to recruit transcriptional machinery to dCas9 and its RNA directed binding...

...determine purity of the AAV preparation. Figure 1: Silver staining of purified and non-purified AAV ...of Pennsylvania Vector Core. Transfections are performed using the transfer plasmid, a plasmid encoding...PCR with primers targeting the transfer plasmid used in the transfection. PCR products are analyzed by ...cell cultures are inspected for sterility. Transducibility Some viral vectors are tested in vitro and ...images).     Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre (55634...filtration through a 0.45 µm polyethersulfone (PES) membrane. The preparations are then aliquoted, frozen, ...from Wang et al., 2018). Briefly, 293T cells are transduced with serial dilutions of a lentiviral vector,...

...antibiotic selection. Essentially, this gene is a 2-in-1 providing both bacterial and eukaryotic selection,...in a plasmid. During the propagation and/or transformation process, some bacteria will either never receive... is nifty because the recovery period after transformation (the dreaded 60 minutes shaking at 37 degrees... available, cost-effective, timesaving for transformations. Cons: Prone to satellite colony formation ..., interchangeable with Amp, timesaving for transformations. Con: Expensive.   Kanamycin Kanamycin, or...selection gene. However, it does require a slower transformation recovery period than amp or carb (60 minutes...confers mammalian resistance to G418. Con: Slow transformation   Spectinomycin/streptomycin Spectinomycin...

... of low-toxicity RNA editing tools.   Fig. 1 Schematic of luciferase RNA knockdown assays using...PRIME, PASTE relies on single-stranded nicks instead of the double-stranded breaks used in other insertion...target. In addition, when they looked at the transformed E. coli expressing Cas7-11, they found no evidence...broadly similar to shRNA and Cas13 systems in a range of mammalian cell lines. However, in those same ... of landing sites via Cas9-directed reverse transcriptases, followed bylanding site recognition and integration...