File:Jr pulsegen 4.jpg

1% agarose gel of PCR products of site directed mutagenesis of the final pulse generator assembly consisting of R0040 I763026 I739001. Template DNA of the pulse generator precursor R0040 I763026 I739001 was amplified by PCR in eight different reactions using primers 276F forward 5’-GGATACGACGATTTTGAAGACAGCTC; T276A forward 5’-GGATACGACGATGCGGAAGACAGCTC; R197F reverse 5’-CAGCCAGCCAGAAACAGACGCGCCG ; R197A reverse 5’-CAGCCAGCCAGCGCCAGACGCGCCG; T276F reverse 5’-GAGCTGTCTTCAAAATCGTCGTATCC ; T276A reverse 5’-GAGCTGTCTTCCGCATCGTCGTATCC in combinations as to produce the desired mutations R197A; R197F; T276A; T276F and double mutants R197A T276A; R197A T276F; R197F T276A; R197F T276F. Each reaction setup contained 35ul water, 10ul Finnzyme Phusion polymerase HF buffer, 1ul 10mM dNTPs, 5ul primer mix (primers at 100ng/ul), 1ul of diluted DNA template (approximate concentration after dilution 10ng/ul), 0.5ul Finnzyme Phusion Hot Start polymerase. The reaction was run with the following thermocycler program: 95C for 30sec, cycle start, 95C 30 sec, 55C 1min, 72C 2min, cycle end, 18 repeats, 72C for 30min, 4C hold. After the PCR reaction had gone to completion, 10ul of 50mM MgCl2 were added to each reaction tube and the contents were thoroughly mixed. 1ul of DpnI (equivalent to 20 units) were added to each tube and the contents were thoroughly mixed. The reaction was incubated in the thermocycler following the program: 37C for 120min, 4C hold. Samples were purified with Qiaquick PCR purification kit and separated on a 1% agarose gel stained with ethidium bromide. It is obvious that the PCR reaction failed in all cases except for reaction number 4, where a small amount of product was generated. Interestingly, amplification between the primers – as evidenced by the bands slightly above 200bp in lanes 5 to 8 – nevertheless took place in the double mutant setup.