A per contra to the elegant #SDM techniques, phosphorylated primers could do wonders in increasing the efficiency of your SDM experiment. Dive in for a #DIY simple technique with all the #buffer compositions!
Principle
The principle here is the #phosphorylation of the 5' ends of the primers, so that when the complete #amplicon with the #mutation is formed, there is an increased probability of formation of the circular #plasmid. This would dramatically increase #efficiency as now, we are not completely dependent on the E. coli #ligase for circularization of the #mutant plasmid.
Method
Primer Phosphorylation (incubate at 37C for 30 mins):
SDM PCR Reaction:
PCR Cycle:
Initial denaturation, 94C: 4 min
In Cycle (16X),
94C: 1 min
{(4X G/C + 2X A/T) -10}C: 1 min
72C: 2.5min/kb + 5min
Final elongation, 72C: elongation time
Hold: as per convenience (4C/ 20C)
DpnI digestion:
As this protocol uses a wild-type/ parent DNA molecule, DpnI treat the PCR product before transformation.
Ligation:
Blunt end ligation of the PCR product ends will increase the efficiency of your mutagenesis manifold. This can be done by incubating the PCR product with T4 DNA ligase.
Transformation.
More to gorge on:
Comments