Team:Heidelberg/Templates/Del week10 FG

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03-07-2013

Amplification from FS_06 to FS_09; 8.5 kb

Amplification of DelA-F and DelF-G run at 130 V, 0.8 % gel (TAE)
Gel after removing the DelA-F-fragment.
Reaction
µl 1st PCR what µl 2nd PCR
1 D. acidovorans DSM-39 1
2.5 FS_06 (1/10) 2.5
2.5 FS_09 (1/10) 2.5
25 Phusion Master Mix 25
- DMSO 2.5
19 dd H2O 19


Conditions
Biorad MyCycler
Cycles-PCR temperature [°C] Time [s]
1 98 10
30 98 1
72 2:30 min
1 72 10 min
1 4 inf

Results:

  • Amplification of DelFG did not work
  • PCR will be repeated with a lower annealing temperature of 68°C (touchdown)

Amplification from FS_06 to FS_09; 8.5 kb

Gel electrophoresis of the PCR products of DelA-F (first three lane (counted from left)) and DelF-G (4th till 6th lane)
Gel after removing DelA-F-fragment
Reaction
what µl 2nd PCR
D. acidovorans DSM-39 1
FS_06 (1/10) 2.5
FS_09 (1/10) 2.5
Phusion Master Mix 25
DMSO 2.5
dd H2O 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 of DelFG did not work, many different side product occured, PCR will be repeated with other primers

Amplification from FS_06 to FS_11; 11.6 kb

Reaction
what µl
D. acidovorans DSM-39 1
FS_06: (1/10) 1
FS_11: (1/10) 1
Phusion Master Mix 10
DMSO 1
dd H2O 6
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

05-07-2013

Amplification from FS_06 to FS_07; 5.2 kb

Reaction
Gel electrophoresis of DelF-G, Backbone, DelF-G (5.1 kb), DelG (6.4 kb).
after Gel excision
what µl
D. acidovorans DSM-39 1
FS_06: (1/10) 1
FS_07: (1/10) 1
Phusion Master Mix 10
DMSO 1
dd H2O 6
Conditions
Biorad MyCycler
Cycles-PCR temperature [°C] Time [s]
1 98 5
14 98 1
62 ↓ 0.5 5
72 3:20 min
16 98 1
60 5
72 3:20 min
1 72 12 min
1 4 inf
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