Team:Heidelberg/Templates/Del week10 AE

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06-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

PCR for amplification of del fragments E-G and A-E, AE+=PCR AE with DMSO, AE-=PCR without DMSO, M= Marker; wanted amplicon sizes are: AE=5.3kb, EG=11.0
Reaction
what µL
D. acidovorans DSM-39 1
FS_02: (1/10) 1
FS_03: (1/10) 1
Phusion Master Mix 10
DMSO 1/-
dd H2O 6/7
PCR for amplification of del fragments AE and EG, cut
Conditions
Biorad MyCycler
Cycles-PCR temperature [°C] Time [s]
1 98 5
12 98 1
66 ↓ 0.5 5
72 2:30 min
18 98 1
63 5
72 2:30 min
1 72 10 min
1 4 inf

Results:

  • Amplification worked with 5% DMSO
  • Repeat Amplification with the same protocol to increase concentration when DNA is extracted from gel slices
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