PCR+Screen+for+SMC1fs+4-19-13

SMC1fs PCR 4-19-13

I've had some trouble with my PCRs with SMC1fs lately. I would like to repeat the SMC1fs PCR using conditions closer to conditions that have worked previously.

Two experiments I have done previously were:

SMC1fs PCR in total 4T1 cDNA 5-2-12 SMC1fs PCR in 4T1 cDNA lysate pools 3-30-12

-5-2-12 experiment { cDNA used previously 4T1 Tumor 2nd Strand cDNA 3-8-11 4T1 in mouse cDNA 9-25

Reaction 5 uL 10X buffer 0.1 uL of 100,000 uM dNTP 1 uL forward primer 1 uL reverse primer 1 uL template 41.4 uL H20 0.5 uL dynazyme

Thermocycle 95C 2m, (95C 30s, 50C 30s, 72C 30s)X30, 72C 5s, 4C hold }

3-30-12 experiment { cDNA used previously 4T1 in mouse cDNA 9/25

Reaction 5 uL 10X dynazyme buffer 0.1 uL of 100,000 uM dNTP 1 uL forward 1 uL reverse 1 uL template 41.4 uL H20 0.5 uL dynazyme

Thermocycle conditions 95 C 2 m, (95C 30s, 50C 30s, 72C 30s)X30, 72C 5m }

See 4-20-13 of notebook for more details

I chose to do a hot start touchdown pcr as well. Thermocycle conditions for this: 95 C 2 m, (95C 30s, variable annealing temp 30s, 72C 30s)X30, 72C 5m

c for cycle c1: Tm of 58.1 c2: 57.1 c3: 56.1 c4: 55.1 c5: 54.1 c6: 53.1 c7: 52.1 c8: 51.1 c9: 50.1 c10: 49.1 c11-36: 48.1