...Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, Smith HO. (2009). Enzymatic assembly of DNA molecules...opens in a new window) Gibson DG, Smith HO, Hutchison CA, Venter JC, Merryman C. (2010). Chemical synthesis...

...A549 cells stably expressing Cas9. A549 cells were transduced with lentiCas9-Blast 1 and then selected with...Figure 2: Cas9 expression in monoclonal cell lines generated from A549 cells transduced with lentiCas9-Blast... express Cas9 and are included as a control (WT). Vinculin is a loading control. 1 lentiCas9-Blast was...additional 3 weeks under blasticidin selection. Anti-Cas9 Western blotting was performed on whole-cell extracts...

...consisting of: 5.0 g yeast extract 10.0 g peptone from casein 10.0 g sodium chloride 12.0 g agar-agar 1 L Sterile...actually make a bit more than 200 mL (~220 mL) just in case we spill anything or have small errors in measurement...the autoclave. Transfer the sterile water (in our case 220 mL) to the same bottle and swirl to form a medium...s manufacturer to make sure this is true in your case. Retrieve your molten agar mix from the autoclave...positive and negative test results. Sample Data In all cases below (-) indicates that the tested strain is not...

...protein concentration in the lysate, running a precast SDS-PAGE gel, and immunoblotting....protein concentration in the lysate, running a precast SDS-PAGE gel, and immunoblotting. Sharing speeds...23227 β-mercaptoethanol 4X protein loading buffer Precast SDS-PAGE gel SDS-PAGE running buffer Prestained...min at 100 °C . Section 3: SDS-PAGE Prepare the precast gel as follows: Remove the gel from the plastic...

...and shift its activity back to polymerase. In our case, dGTP will be used in the reaction, meaning that...there are no gaps in the final assembly. In this case, 15 bp of homologous sequence is used, plus a minimum...following the manufacturer's instructions. In this case, we will include the free nucleotide dGTP in the...

...seeded the day before transduction. Note that in that case, polybrene should only be added at the time of viral...sensitive to lentivirus or to polybrene, in which case this incubation step can be reduced to 8 h and the...serial dilutions of the lentiviral vector pHAGE EF1α dCas9-KRAB . The following day, cells were treated with...

...A549 cells stably expressing Cas9. A549 cells were transduced with lentiCas9-Blast and then selected with...formed by expansion of single cells for 13 days. lentiCas9-Blast was a gift from Feng Zhang (Addgene plasmid...

... how to perform gel electrophoresis. Equipment Casting tray Well combs Voltage source Gel box UV light...prefer to microwave in pulses, swirling the flask occasionally as the solution heats up.). Caution HOT! Be ...

...of a hairpin structure within your primer. In our case, we will add TAAGCA, resulting in a final Forward...to the sequence to be amplified (the ORF in this case), not the Tm of the entire primer. If you are amplifying...

...antibody concentration may have been too low. In this case, use higher antibody concentrations. If the reaction...antibody concentration may have been too high. In this case, use lower antibody concentrations. The ideal range...

...light that your substance maximally absorbs. In the case of nucleic acid (DNA and RNA), the maximal absorbance...

... deletions in mammalian cells lines using CRISPR/Cas9 Genomic Deletions with CRISPR Protocol Over-Agar...

... not dislodge any pellets or re-mix liquids. In cases where you are separating solids from a liquid, pellets...

...transduced with a range of dilutions of pHAGE-TO-dCas9-3XmCherry . 72 h post transduction, cells were assayed...

...per 10 cm dish). Using transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid DNA 27.8 μg), this would...

... or can come preloaded into tip boxes. In either case, once assembled into their boxes, pipette tips can...

...chamber. Use the metal spatula to gently break the gel cast open. Use a razor blade to cut the top of the wells...

... Prepare one to two more conical tubes on ice in case you need to spread out the harvested cells further...

...the stuffer. The AgeI site is destroyed in most cases (depending on the target sequence), while the EcoRI...