...targeting the region where the edit should be made HDR requires a repair template and displays reduced efficiency...

...toward expanding this capacity. Traditionally, AAV requires the presence of another "helper" virus, such as...

...crRNA/gRNA length gRNA: ~100 nt crNA: ~42 nt Requires tracrRNA? Yes No dsDNA cleavage Blunt end 5'...vector expresses Cas9 and up to 7 gRNAs Each gRNA requires its own promoter Sakuma et al   Multiplexed...expresses Cas9, eGFP, and up to 4 gRNAs Each gRNA requires its own promoter   Kabadi et al Csy4-Cleavable...gRNAs expressed from a polycistronic transcript  Requires co-transfection of Cas9 Nissim et alTsai et al...gRNAs expressed from a polycistronic transcript Requires co-transfection of Cas9   Xie et al  Multiplexing...

...binding sites, commonly used in Drosophila Specific Requires the presence of Gal4 gene to activate promoter...Promoter from T7 bacteriophage Constitutive, but requires T7 RNA polymerase. When used for in vitro transcription... Negligible basal expression when not induced. Requires T7 RNA polymerase, which is also controlled by...Promoter from Sp6 bacteriophage Constitutive, but requires SP6 RNA polymerase. SP6 polymerase has a high ...Promoter from T3 bacteriophage Constitutive, but requires T3 RNA polymerase When used for in vitro transcription...

...materials. Problems with Chip-seq ChIP-seq typically requires a large amount of input material, cells or tissue...experiment can be a major technical hurdle.   ChIP-seq requires  cross-linking during an initial fixation step...wing as a model. A typical ChIP-Seq experiment requires around 1,000 wings. Collecting that much tissue...controlled by the addition of calcium, which MNase requires for its nuclease activity and is chelated from...

...select first hires who are well-suited, productive, and fit well with your goals. These first hires will set...set the tone and atmosphere that future hires will look up to." She also recommends to seek out advice...lab and selectively picking the first couple of hires to build a strong, collaborative team. And take ...

...three lab heads how they go about selecting new hires. This is followed by some perhaps surprising, yet...scientists for their labs. Tom Ellis selects new hires from a pool of CVs collected from throughout the...are dedicated and interested in science. “Postdoc hires are simpler because of their documented history ...with their interests.” George Church selects new hires initially based on those who notice and embrace ...

...seem like the easier of the two, but it actually requires more technical skill to place the needle in the...the vertebrae, so hitting the correct location requires a lot of skill and anatomical knowledge. Intrathecal... the lungs. It is an advanced technique, as it requires intubation of the mice, which can be tricky given... intranasal is easier, less invasive, and only requires a pipette. Since the substances are administered...

...preps, lentiviral titration requires an additional couple of days, as it requires transduction of more cells...companies. Production of both AAV and lentivirus requires three plasmids — a transfer plasmid containing...baculovirus plasmid. Production of baculovirus requires only two plasmids — your transfer plasmid and ...

...AAV replication uses the host’s polymerase, but requires Rep proteins to process a double-stranded intermediate...AAV than I think I need. Unlike lentivirus, AAV requires a higher number of viral particles for efficient...several hours on a heated stir plate to dissolve and requires monitoring so that the solution doesn’t get too...AAV for my cells of interest. Every batch of AAV requires its own MOI optimization to account for batch-...

... of hard-to-achieve factors. For example, FRET requires that fusion proteins be in close proximity and...FRET, as the overexpression of fusion proteins it requires can lead to aberrant subcellular localization ... construction Fusing FP fragments to your PIPs requires careful selection of FP fragments and, preferably...information, constructing appropriate controls may requires that you empirically test a number of mutants ...

...conjugate, and changing your reporter molecule requires either re-conjugating or re-purchasing your primary...most protein from your gel to your membrane. It requires anywhere from sixty minutes to overnight to run... can be done.  Note that each type of transfer requires different equipment. Wet transfers can be done...compatible with chemiluminescence reporters and requires access to a darkroom and a developer. Film is ...exposures for a blot with varying signals on it. It requires long exposure times (up to thirty minutes!) to...

...myristoylation requires an N-terminal consensus sequence (MG-) and prenylation requires a C-terminal consensus... 2002). GFP needs oxygen Chromophore formation requires a fully folded beta-barrel structure, followed...chromophore (Tsien, 1998). Importantly, this reaction requires molecular oxygen and as a consequence GFP remains...

...mutations. Ultimately, successful allelic exchange requires two sequential homologous recombination events...events, but because these events are rare, each requires selection. These two steps are (Figure 1): The ...and excision of the vector backbone. This step requires “counter-selection,” meaning that the selection...generate precise, unmarked mutants, but it frequently requires considerable optimization to ensure strong counter-selection...

...James M. Wilson AV-9-PV0109 105533-AAV9 pAAV.CMV.Luc.IRES.EGFP.SV40 James M. Wilson AV-9-PV1262 Plasmid not...

...promoters for transcriptional regulation, so it requires a different approach for regulating transgene ...Generating iPSCs with plasmid-based expression requires serial transfection of 1 or 2 plasmids that express... the gold standard instead. Teratoma formation requires injecting iPSCs into immunodeficient mice and ...suited for these situations. The second scenario requires higher levels of safety and as little chance of...

...182653 pcDNA3.1(+)_U6 tRNAPyl_CMV NESPylRS(AF)_IRES_eRF1(E55D)-HA Y306A/Y384F (AF) pyrrolysine (Pyl) tRNA...

...must be performed after primary purification and requires a second clean up step to remove the cleaved tag...Also, purifying GST by affinity chromatography requires proper folding of the tag, which prevents insoluble.... Mammalian purification is still possible but requires optimization and often more stringent washing....

... endeavor, NEBuilder requires upfront planning. This cloning methodology requires that the fragments to...simplify this process by using NEBuilder which requires only a single-stranded synthetic oligo to generate...

...frequency. Although highly useful, this technique requires extensive sample processing to avoid disrupting...appear red. To enable this color switching, TRICK requires the following components: a GFP protein that ...caveats. In both cases, creating a reporter mRNA requires inserting multiple hairpin sequences into the ...