Designing and Generating CRISPR-Cas Mutants: Difference between revisions
Added cloning information |
updated annealing conditions |
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** Digest for 1h in, gel purify the fragment from an agarose gel. | ** Digest for 1h in, gel purify the fragment from an agarose gel. | ||
* Next prepare the insert by annealing and phosphorylating the primers in a PCR tube: | * Next prepare the insert by annealing and phosphorylating the primers in a PCR tube: | ||
** | ** 1 uL of a 100 uM stock of each oligo with 4.5 uL of water | ||
** 2.5 uL of ligase buffer | |||
** 1 uL of T4 PNK | |||
** | ** Incubate at 37C for 30 mins then 95C for 5 mins to heat inactivate PNK then ramp down to 25C at 5C/min to allow the oligos to anneal | ||
* Combine the ligation mixture in an eppendorf tube: | * Combine the ligation mixture in an eppendorf tube: | ||
** | ** 5 ng of vector (~10 pmoles of vector) | ||
** | ** 3 uL of annealed insert (~30 pmoles of Insert) or water as a blank | ||
** 2.5 uL of 4X ligation buffer | ** 2.5 uL of 4X ligation buffer | ||
** 1 uL of T4 DNA Ligase | ** 1 uL of T4 DNA Ligase | ||