PCR Screen for SMC1fs 4-19-13

2015-01-13

azim58 - 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

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