'Round-the-horn site-directed mutagenesis
‘Round the Horn Site-directed mutagenesis
Forward primer has intended mutation at its 5’ end. Design such that the annealing portion has Tm ~60-63º.
Reverse primer sits on the complementary strand and abuts the site of mutation.
If the change needed is large, both primers can have mutant 5’ ends. Primers without mutations can be spaced with a gap between them to make deletions.
Primer stock at 100 M in water or TE. For each primer:
37 L water 5 L 10 Kinase reaction buffer (comes with PNK) 1 L 50 mM MgSO4 (comes with most polymerases) 5 L primer (10 M final) 1 L 100 mM ATP 1 L PNK 50
37 degrees for 30-60 minutes. Heat each reaction to kill the PNK (65 degrees for 20 minutes, or 95 degrees for 5 min)
PCR Set up on ice. After mixing, put into pre-heated PCR block.
39 L water 5 L 10X polymerase buffer 1.5 L forward primer (0.3 M final) 1.5 L reverse primer 1 L dNTPs (20 mM each) 1 L template (mini-prep’d plasmid) 1 L polymerase (not Taq, use Vent, Deep Vent, 9°N, Pfu) 50 L
(1) 95º 1’ (2) 93º 30” (3) 55º 30” (~5º below Tm) (4) 72º 2’ per kb being amplified (5) Goto (2) 27 more times (6) 4º hold
Run 5 L on a gel. I put EtBr in the gel so I can check to see if there is a product within a few minutes. If you see the band, digest the template in the PCR reactions by adding 1 L of DpnI, mix, and incubate for at least 60 minutes at 37º.
Run a preparative gel of the PCR product, excise the band, and use a kit to extract the product.
2.3 L water* 2 L PCR product 0.5 L 10X ligation buffer 0.2 L ligase 5 L
- If there is not much product, replace the water fraction with more PCR product.
Incubate over night at room temp.
Works best with electroporation or fresh TSS cells. Transform 4 L into 50 L cells. Plate all of it.