Team:Heidelberg/Templates/Del week14 AG

From 2013.igem.org

30-07-2013

Amplification from FS_02 to FS_26; 16.7 kb

Amplification of Del A-G under different conditions and DelAF; run at 100 V 0.8% agarose gel (TAE)

4 reactions, 68°C Touchdown and 65°C constant with 200nM and 400nM Primers

Reaction
what µl
D. acidovorans DSM-39 1
FS_02: (1/10) 2/4
FS_26: (1/10) 2/4
Phusion flash Master Mix 10
DMSO 1
dd H2O 0/4
Conditions I
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
65 5
72 5:40
1 72 12 min
1 10 inf


Conditions II
Biometra TProfessional Basic
Cycles-PCR temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 5:40 min
18 98 1
66 5
72 5:40 min
1 72 10 min
1 4 inf

Results:

  • Amplification of DelAG did not work, neither annealing at 65°C constant nor a touchdown starting from 68°C led to the desired product
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