Team:Heidelberg/Templates/Del week12 G

From 2013.igem.org

Contents

16-07-2013

Amplification from FS_08 to FS_11_short; 6.5 kb

Gel for testing concentrations of older fragments; run at 100 V, 0.8 % gel (TAE)
Gel for testing concentrations of fragment DelG (16.07; Conditions II; after gel extraction; run at 100 V, 0.8 % gel (TAE)


Reaction
what µl
D. acidovorans DSM-39 1
FS_08: (1/10) 2
FS_11_short: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4


Conditions I
Biorad C1000 Touch Block A
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 2:30 min
24 98 1
63 5
72 2:30 min
1 72 10 min
1 12 inf


Conditions II
Biorad C1000 Touch Block B
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 2:30 min
18 98 1
66 5
72 2:30 min
1 72 10 min
1 12 inf


Conditions III
Biometra T100
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
70 ↓ 0.5 5
72 2:30 min
18 98 1
68 5
72 2:30 min
1 72 10 min
1 12 inf

Results:

  • Only a small band with conditions II was visible
  • There were no bands with the other two
  • Bands were cut out and DNA purified using QIAquick Gel Extration Kit.

18-07-2013

Amplification from FS_08 to FS_11_short; 6.5 kb

lane2=Re-PCR DelG, lane3=amplification DelG (18.07), rest see DelH
Reaction
what µl
D. acidovorans DSM-39 1
FS_08: (1/10) 2
FS_11_short: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4


Conditions
Biorad C1000 Touch
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
64 ↓ 0.5 5
72 2:10 min
18 98 1
60 5
72 2:10 min
1 72 10 min
1 12 inf

Results:

  • Amplification of DelG did not work, no product was detectable
  • PCR will be repeated at higher temperature to decrease formation of secondary structures and thereby improve binding of primers to the desired sequence

Re-Amplification from FS_08 to FS_11_short; 6.5 kb; 09-07-2013)

lane2=Re-PCR DelG, lane3=amplification DelG (18.07), rest see DelH
Reaction
what µl
DelG (09-07-2013) 4
FS_08 (1/10) 2
FS_11_short (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 1
Conditions II
Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0.5 5
72 2:30
16 98 1
63 5
72 2:30
1 72 10min
1 4 inf

Results:

  • Amplification of DelG was not sucessful, the Re-PCR did not yield the desired product
  • forward Primer will be reused but reverse primer changed, in order to obtain a shorter amplicon and another strategy for the Gibson Assembly

19-07-2013

Amplification from FS_08 to FS_09; 3.3 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_08: (1/10) 2
FS_09: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4
Conditions
Biorad MyCycler
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
70 ↓ 0.5 5
72 1:00
18 98 1
66 5
72 1:00
1 72 6min
1 12 inf

Results:

  • Amplification of DelG did not lead to the desired product, a small band of the desired size was visible and cut for validation and Re-PCR
  • Re-PCR will be run

21-07-2013

Re-PCR from FS_08 to FS_09; 3.3 kb; 19-07-2013

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) cut2; run at 100 V, 0.8 % gel (TAE)
Reaction
what µl
Fragment FS_08 to FS_09 (19-07-2013) 1
FS_08: (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
70 ↓ 0.5 5
72 1:00
18 98 1
66 5
72 1:00
1 72 6min
1 12 inf

Results:

  • Re-Amplification of DelG did not work
  • initial amplification will be repeated at lower annealing temperature
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