Team:Heidelberg/Templates/Del week12 AE

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Contents

19-07-2013

Amplificaction from FS_02 to FS_03; 5.3 kb

+=with DMSO, -=without DMSO; expected amplicon sizes: LP=6.4kb, 8-9=3.1kb, 21-09 and 20-09=8.1kb, 22-13s=2.6kb, AE=5.3kb; run at 100 V, 0.8 % gel (TAE)
Amplifications of 18.07 and 19.07, cut
Reaction

2x ~50 µL

what µL
D. acidovorans DSM-39 1
FS_02: (1/10) 2.5
FS_03: (1/10) 2.5
Phusion flash Master Mix 25
DMSO 2.5
dd H2O 19
Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 5
12 98 1
66 ↓ 0.5 5
72 2:30
18 98 1
63 5
72 2:30
1 72 10min
1 12 inf

Results:

  • Amplification of DelAE did not work since a different cycler was used

20-07-2013

Amplification from FS_02 to FS_03; 5.3 kb

Amplification of AE, OP, FG (20.07); expected amplicon sizes: 21-09(FG)=8.1kb, 22-13s(OP)=2.6kb, AE=5.3kb; run at 100 V, 0.8 % gel (TAE)
Reaction
what µL
D. acidovorans DSM-39 1
FS_02: (1/10) 2.5
FS_03: (1/10) 2.5
Phusion flash Master Mix 25
DMSO 2,5
dd H2O 19
Conditions
Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 5
12 98 1
66 ↓ 0.5 5
72 2:30
18 98 1
63 5
72 2:30
1 72 10min
1 12 inf

Results:

  • Amplification of DelAE worked but a smear occured, therefore bands were cut out carefully and only used for a test restriction digest
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