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Team:Paris Bettencourt/Notebook/Trojan Horse/Thursday 1st August.html
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Revision as of 08:40, 3 October 2013
Trojan Horse
ASDF1st August
Aude & Vincent
Cloning Kanamycin resistance gene into pACYCDuet-1:
-PCR of Kanamycin resistance gene from pCOLADuet-1 with primer iT.0003 and iT.0004
-electrophoresis of PCR product: 1 band between 900 and 1000bp: coherent with KanR length
-cutting KanR bands:
-digestion with ECOR1 and Xho1:
PCR product digest for cloning
|
Reagent |
Volume |
|
Purified PCR Product |
16 ul |
|
H2O |
0 ul |
|
10x FastDigest Buffer |
2 ul |
|
FastDigest Enzyme 1 |
1 ul |
|
FastDigest Enzyme 2 |
1 ul |
|
Total Volume |
20.0 ul |
Digest for 0.5 - 1 hours at 37 °C with shaking.
-digestion of pACYCDuet-1 with ECOR1 and Xho1:
Vector backbone digest for cloning
|
Reagent |
Volume |
|
Purified Plasmid |
20 ul |
|
H2O |
65 ul |
|
10x FastDigest Buffer |
10 ul |
|
FastDigest Enzyme 1 |
2.5 ul |
|
FastDigest Enzyme 2 |
2.5 ul |
|
FastAP Phosphatase |
0 ul |
|
Total Volume |
100.0 ul |
Digest for 2 - 4 hours at 37 °C with shaking.
-electrophoresis of pACYCDuet-1 digestion products:
column 1: 1kb plus marker
column 2 & 3: digestion product
column 4: undigested pACYCDuet-1
nothing appear on gel after digestion, therefore we will miniprep pACYCDuet-1 and try again to digest it.
