Team:Heidelberg/Templates/Del week14 AF

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Contents

29-07-2013

Restriction digest of fragment FS_02 to FS_05; 11.2 kb; 27-07-2013 with BglII

Test restriction digest (29.07); run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for about 3 hours

what µL
FS_02 to FS_05 (27-07-2013) 15
BglII 1
Buffer 3.1 2
dd H2O 2
Expected fragment lengths [bp] 7229, 2146, 1862

Results:

  • Test restriction digest worked out as predicted

30-07-2013

Amplification from FS_02 to FS_05; 11.2 kb

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

2 reactions 200nM/400nM Primers

Reaction
what µL 2nd PCR
D. acidovorans DSM-39 1
FS_02 (1/10) 5/2.5
FS_05 (1/10) 5/2.5
Phusion Master Mix 25
DMSO 2.5
dd H2O 11.5/16.5
Conditions
Biorad MyCycler*
Cycles-PCR temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 3 min
18 98 1
66 5
72 3 min
1 72 10 min
1 4 inf

Results:

  • Amplification did not work properly, as there were several unspecific bands. However we still had product.
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