Team:Heidelberg/Templates/Del week12 FG

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Contents

15-07-2013

Re-PCR for amplification of DelFG (15.07) run at 100 V, 0.8 % gel (TAE)


Re-Amplification from FS_06 to FS_09; 8.5 kb; 13-07-2013)

Reaction
what µl
DelEG (FS06-FS09) 1
FS_06: (1/10) 2
FS_09: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions

Cycler incubation room right

Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72 3:00 min
1 72 10 min
1 12 inf

Results:

  • Amplification of DelFG did not work
  • two unecpected bands appeared like in the previous trials

19-07-2013

Amplification from FS_20 to FS_09; 8.5 kb

+=with DMSO, -=without DMSO; expected amplicon sizes: LP=6.4kbp, 8-9=3.1kbp, 21-09 and 20-09=8.1kbp, 22-13s=2.6kbp, AE=5.3kbp; run at 100 V, 0.8 % gel (TAE)
Amplifications of 18.07 and 19.07, cut
Reaction
what µl
D. acidovorans DSM-39 1
FS_20: (1/10) 2
FS_09: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 2:50
18 98 1
66 5
72 2:50
1 72 10min
1 12 inf

Results:

  • Amplification of DelFG did not work with FS_20 to FS_09
  • as PCR worked with a different set of Primers beeing FS_21 to FS_09, see below, the PCR for this primer combination will be optimized instead of using FS_20 to FS_09

Amplification from FS_21 to FS_09; 8.5 kb

Reaction
what µl
D. acidovorans DSM-39 1
FS_21: (1/10) 2
FS_09: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions

-->New cycler (not two block)

Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 2:50
18 98 1
66 5
72 2:50
1 72 10min
1 12 inf

Results:

  • Amplification worked with FS_21 to FS_09 but not with FS_20 to FS_09
  • band was cut out and DNA purified using QIAquick Gel Extraction Kit
  • PCR will be repeated to get rid of side product at about 1.5 kb as well as smear, therefore annealing temperature will be increased

20-07-2013

Amplification from FS_21 to FS_09; 8.5 kb

Amplification of AE, OP, FG (20.07); expected amplicon sizes: 21-09(FG)=8.1kbp, 22-13s(OP)=2.6kbp, AE=5.3kbp; run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
D. acidovorans DSM-39 1
FS_21: (1/10) 2
FS_09: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad C1000 Touch Block A
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
70 ↓ 0.5 5
72 2:50
18 98 1
68 5
72 2:50
1 72 10min
1 12 inf

Results:

  • Amplification of DelFG did not work only an uneqxpected band a smear occured, but the intended product was not detectable
  • other primer combinations might be tried or PCR has to be repeated with the previous conditions not leading to an optimal product quality

21-07-2013

Amplification from FS_20 to FS_07; 5.2 kb

Amplification of FS20-FS07 (FG), Re-PCRs FS08-FS09 (G), FS22-13_short (OP) and FS04-FS07 (all 21.07); - without DMSO, + with DMSO; run at 100 V, 0.8 % gel (TAE)
Amplification of FS20-FS07 (FG), Re-PCRs FS08-FS09 (G), FS22-13_short (OP) and FS04-FS07 (all 21.07) cut1; run at 100 V, 0.8 % gel (TAE)
Amplification of FS20-FS07 (FG), Re-PCRs FS08-FS09 (G), FS22-13_short (OP) and FS04-FS07 (all 21.07) cut2; run at 100 V, 0.8 % gel (TAE)

2x 20µl (with, without DMSO)

Reaction
what µl
D. acidovorans DSM-39 1
FS_20: (1/10) 2
FS_07: (1/10) 2
Phusion flash Master Mix 10
DMSO 1/-
dd H2O 4/5


Conditions
Biorad C1000 Block A
Cycles temperature [°C] Time [s]
1 98 5
12 98 1
68 ↓ 0.5 5
72 2:00
18 98 1
63 5
72 2:00
1 72 10min
1 12 inf

Results:

  • Amplification of DelFG did not work perfectly, neither with nor without 5% DMSO, nevertheless band of expected size was cut out carefully to be used for a Re-PCR



Re-PCR from FS_21 to FS_09; 8.1kb; 19-07-2013)

Gel for testing concentrations of fragment FS04 to FS07 (14.07 and 15.07), FS_21 to FS_09 (19.07); run at 100 V, 0.8 % gel (TAE)
Amplification of DelOP and Re-PCR of DelFG (21.07); run at 100 V, 0.8 % gel (TAE)
Amplification of DelOP and Re-PCR of DelFG (21.07) cut; run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
Gel extracted fragments FS_21 to FS_09 (19-07-2013) 2
FS_21: (1/10) 2
FS_09: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 3
Conditions
Biorad T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 2:50
18 98 1
66 5
72 2:50
1 72 10min
1 12 inf

Results:

  • Amplification of DelFG did not work with primers FS_21 to FS_09
  • PCR will be repeated with different primers


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