906
edits
Changes
updated annealing conditions
** 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:
** ADD ANNEALING INFO** Run on ANNEALING CYCLE1 uL of a 100 uM stock of each oligo with 4.5 uL of water** Phosphorylate the primers by adding:*** X 2.5 uL of ligase buffer*** 1 uL of T4 PNK*** Incubate at 37C for 30 mins then 60C 95C for 20 5 mins to heat inactivate PNKthen ramp down to 25C at 5C/min to allow the oligos to anneal
* Combine the ligation mixture in an eppendorf tube:
** 50 5 ng of vector (~100 10 pmoles of vector)** X 3 uL of annealed insert (~300 30 pmoles of Insert)or water as a blank
** 2.5 uL of 4X ligation buffer
** 1 uL of T4 DNA Ligase