A A | B B | C C | D D | E E | F F | G G | |
1 1 | Component | 50 ul reaction | Final concentration | Thermocycling conditions | |||
2 2 | NS-free H2O | 32.25 | STEP | TEMP | TIME | ||
3 3 | 5X Phusion HF Buffer | 10 | 1X | Initial Denaturation | 98C | 30s | |
4 4 | 10 mM dNTPs | 1 | 200 uM | 30 cycles | 98C | 15s | |
5 5 | 2.5 | 0.25 uM | 60C | 30s | |||
6 6 | 2.5 | 0.25 uM | 72C | 30s | |||
7 7 | 1.25 | 1.25 ng/ul | Final extension | 72C | 5 minutes | ||
8 8 | Phusion DNA Polymerase | 0.5 | 1.0 units/50 ul PCR | Hold | 4C | forever | |
9 9 | Total | 50 |
NB: Wipe vacuum centrifuge clean with 70 % ethanol and concentrate samples until <15µL. It is not a problem if you end up drying the DNA as it will be resuspended subsequently. Usually, it takes about 5 minutes to evaporate 15µL of TE/H2O at 60C. Check volume every 5 minutes to prevent overdrying (although your experiment should still work once you resuspend the DNA in your nucleofection mix; I tried when I accidentally overdried some samples).
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This protocol describes a cloning-free gBlocks method for successful CRISPR-mediated gene editing in cell culture. The protocol is a modification of the cloning-free CRISPR method published by Arbab et al. (Stem Cell Reports 2015).