Team:Heidelberg/Templates/Del week13 AF

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Revision as of 22:52, 29 September 2013

Contents

26-07-2013

Restriction digest of FS_02 to FS_05; 11.2 kb; 04-07-2013 with EcoRI-HF

File:20130726 2,5µllog2 AEEcoRIHF AFEcoRIHF GBGlII OPEcoRIHFbesch.png
Restriction digest of DelAE (FS_02 to FS_03 ;08.07), DelAF (FS_02 to FS_05; 04.07) and DelOP (FS_22 to FS_13short ;21.07) with EcoRI-HF and digest of DelG (FS_08 to FS_23 cut1 ;23.07) with BglII; run at 100 V, 0.8 % gel (TAE)


Incubation at 37°C for 1hour 45min

what µL
FS_02 to FS_05 (04.07) 15
EcoRI-HF 0.5
Buffer CutSmart 2
dd H2O 2

Results:

  • restriction digest did not work as expected
  • test restriction will be repeat with higher amount of enzyme and longer incubation time

27-07-2013

Amplification of FS_02 to FS_05; 11.2 kb

File:20130727 2log 2xAE 2xAF 1kb 2xIliabesch.png
Amplification of DelAE (FS02 to FS03; 27.07,1), Amplification of DelAF (FS_02 to FS05; 27.07); run at 100 V, 0.8 % gel (TAE)
File:20130727 2log 2xAE 2xAF 1kb 2xIliacut.png
Amplification of DelAE (FS02 to FS03; 27.07,1), Amplification of DelAF (FS_02 to FS05; 27.07), cut; run at 100 V, 0.8 % gel (TAE)
Reaction
what µL
D. acidovorans 1
FS_02: (1/10) 2.5
FS_05: (1/10) 2.5
Phusion flash Master Mix 25
DMSO 2.5
dd H2O 16.5
Conditions
Biorad MyCycler*
Cycles temperature [°C] Time [s]
1 98 10
12 98 1
68 ↓ 0,5 5
72 3:00
18 98 1
66 5
72 3:00
1 72 10 min
1 4 inf

Results:

  • Amplification of DelAE worked, but a slight smear as well as several unexpected bands occured
  • bands were cut out and DNA purified using QIAquick Gel Extraction Kit but PCR has to be further optimized in order to improve product quality
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