Site directed mutagenesis

Materials

DNA template, 100 – 200 ng/μl
2 mutagenesis primers, 10 μM, containing desired point mutation as well as silent mutation(s) introducing a novel restriction site
20 mM dNTPs 10x Taq/Pfu buffer
Pfu DNA polymerases

Procedure

• Prepare the PCR reaction mix according to the hot start procedure. Include one negative control sample without Pfu polymerase (incubate sample at the lowest annealing temperature of your gradient)
• Perform gradient PCR using the following thermal cycling conditions:

Step	Temperature °C	Time	No. of cycles
Initial denauturation	94	1-3 min	1
Hot Start / add enzyme	94	pause	1
Denauturation	94	20 sec	20
Annealing	51, 55, 59, 63	20 sec
Extension	72	1 min/500 bp
Final extension	72	10 min	1

• DpnI digestion: prior to DpnI digestion, separate 15 μl PCR product of negative control (will serve as positive control). Add 2 μl DpnI to every PCR sample. Incubate for 2 hours at 37 °C
• Inactivation of DpnI: incubate the digested PCR products at 70 °C for 10 min
• Perform transformation of E. coli cells with the PCR products: 10 μl PCR products (digested or undigested) for 90 μl E. coli competent cells
• Check positive/negative controls. If negative control shows no or low background, check out the mutagenesis samples: starting with the highest gradient temperature, pick every colony and streak out on fresh agar plate (cake style). Next day, isolate the plasmid from the first 12 - 24 clones plate using Birnboim- Dooley protocol
• Restriction analysis: digest the plasmid with the newly incorporated restriction enzyme. Check the size of fragments on the electrophoresis gel compared to the original (wildtype) version.
• Sequence one positive clone and save in strain collection, if correct