906
edits
Changes
Added cloning information
* To make the reverse primer reverse transcribe the copied sequence (removing the NGG and the end, and not including the CACC at the other end). Add AAAC to the 5' end of that reverse transcribed sequence to make the appropriate overhang.
* Name the primers as follows: '''mm-Pygm-gDNA-62-FWD''' where mm = species; Pygm = your gene name, 62 = where this nickase cuts, and FWD or REV is whether the primer is forward or reverse.
===Cloning===
* First digest pX335 vector by adding to a PCR tube:
** 1 ug of pX335,
** 1uL of 10X NEB Buffer 2.1
** 1 uL of CIAP
** 1 uL of BbsI
** water up to 10 uL
** 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 CYCLE
** Phosphorylate the primers by adding:
*** X uL of ligase buffer
*** 1 uL of T4 PNK
*** Incubate at 37C for 30 mins then 60C for 20 mins to heat inactivate PNK
* Combine the ligation mixture in an eppendorf tube:
** 50 ng of vector (~100 pmoles of vector)
** X uL of insert (~300 pmoles of Insert)
** 2.5 uL of 4X ligation buffer
** 1 uL of T4 DNA Ligase
** Water to 10 uL
* Incubate for 10 min at RT
* Transform into competent cells (see [[Transformation of Bacteria]])