Team:Heidelberg/Templates/Del week13 AF

From 2013.igem.org

(Difference between revisions)
(→Restriction digest of FS_02 to FS_05; 11.2 kb; 04-07-2013 with EcoRI-HF)
(→Restriction digest of FS_02 to FS_05; 11.2 kb; 04-07-2013 with EcoRI-HF)
 
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! what !!  µL   
! what !!  µL   
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| FS_02 to FS_05 (04.07) || 15
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| FS_02 to FS_05 (04-07-2013) || 15
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| EcoRI-HF || 0.5
| EcoRI-HF || 0.5

Latest revision as of 18:35, 3 October 2013

Contents

26-07-2013

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

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 (04-07-2013) 15
EcoRI-HF 0.5
Buffer CutSmart 2
dd H2O 2
Expected fragment lengths [bp] 4624, 4354, 2260

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

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)
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 DSM-39 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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