Team:Heidelberg/Templates/Del week12 EG

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Contents

15-07-2013

Amplification from FS_04 to FS_07, 11.1 kb

PCR for amplification of DelEG (FS04-FS07, 15.07), concentration AF(FS2 to FS5; 04.07); desired amplicon size Del EG=11kbp; run at 100 V, 0.8 % gel (TAE)
PCR for amplification of DelEG (FS04-FS07, 15.07) after excision; run at 100 V, 0.8 % gel (TAE)

3x 20µl of reaction mix

Reaction
what µl
D. acidovorans DSM-39 1
FS_04: (1/10) 2
FS_07: (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 3:00 min
18 98 1
68 5
72 3:00 min
1 72 10 min
1 12 inf

Results:

  • Amplification of DelEG worked as expected
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit


21-07-2013

Re-PCR of DelEG (FS_04 to FS_07; 11.1 kb; 05-07-2013)

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 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)
Reaction
what µl
Fragment FS_04 to FS_07 amplified 15-07-2013 1
FS_04: (1/10) 2
FS_07: (1/10) 2
Phusion flash Master Mix 10
DMSO 1
dd H2O 4


Conditions
Biometra TProfessional Basic
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
70 ↓ 0.5 5
72 3:00 min
18 98 1
68 5
72 3:00 min
1 72 10 min
1 12 inf

Results:

  • Re-PCR of DelEG in order to increase specifity did not work


24-07-2013

Restriction digest of DelEG(FS_04 to FS_07; 11.1 kb) with PvuI-HF

Test restriction digest of DelEG FS04 to FS07 (24.07); run at 100 V, 0.8 % gel (TAE)

Incubation at 37°C for 45 min

what µl
FS_04 to FS_07 (14-07-2013 and 15-07-2013) 15
PvuI-HF 0.8
Buffer CutSmart 2
dd H2O 2.8

Results:

  • restriction digest did not work
  • digest will be repeated with newly amplified and purified DelEG


Retrieved from "http://2013.igem.org/Team:Heidelberg/Templates/Del_week12_EG"