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Team:Paris Bettencourt/Notebook/Trojan Horse/Wednesday 14th August.html
From 2013.igem.org
Trojan Horse
ASDF14th August
Place your twit here
PCR of pUC18 (pT007) with iT0007 & iT0008 / PCR of litmus28i-GFP (pT005) with iT0005 & iT0006 (Aude):
Gel Purif (Aude)
Using QiaGen Kit
3 tubes (combined the band)
1 : 13,2 ng/uL
2 : 14 ng/uL
3 : 2O ng/uL
Digestions ( Aude )
Digestion pACY par Xho EcoRI (pour le clonage de Kan dans ACY)
|
Reagent |
Volume |
|
Purified Plasmid |
10 ul |
|
H2O |
75 ul |
|
10x FastDigest Green Buffer |
10 ul |
|
EcoRI |
2.5 ul |
|
XhoI |
2.5 ul |
|
FastAP Phosphatase |
0 ul |
|
Total Volume |
100.0 ul |
Digestion pACY par NcoI BgIII (pour le clonage de lacZalpha dans ACY)
|
Reagent |
Volume |
|
Purified Plasmid |
15 ul |
|
H2O |
75 ul |
|
10x FastDigest Green Buffer |
10 ul |
|
EcoRI |
2.5 ul |
|
XhoI |
2.5 ul |
|
FastAP Phosphatase |
0 ul |
|
Total Volume |
100.0 ul |
=> 2h30 à 37°C
-
Run on a a gel.
-
only see a smear, Everything was digested ,
-
redigest but just 25 minutes as said on the thermoscientific protoc.
Digestions ( CLovis )
-
Digestion pACY par Xho EcoRI (pour le clonage de Kan dans ACY)
|
Reagent |
Volume |
|
Purified Plasmid |
10 ul |
|
H2O |
75 ul |
|
10x FastDigest Green Buffer |
10 ul |
|
EcoRI |
2.5 ul |
|
XhoI |
2.5 ul |
|
FastAP Phosphatase |
0 ul |
|
Total Volume |
100.0 ul |
-
Digestion pACY par NcoI BgIII (pour le clonage de lacZalpha dans ACY)
|
Reagent |
Volume |
|
Purified Plasmid |
15 ul |
|
H2O |
75 ul |
|
10x FastDigest Green Buffer |
10 ul |
|
EcoRI |
2.5 ul |
|
XhoI |
2.5 ul |
|
FastAP Phosphatase |
0 ul |
|
Total Volume |
100.0 ul |
25 minutes at 37°C
It worked!
Gel purification (Clovis)
ACY Nco Dig : 6,3 ng/ul
ACY Xho Dig : 7,8 ng/uL
PCR Gradient of 8 tubes +10°C around 54°C for both PCRs (Litmus18i-GFP) but start not from the miniprep ut from a fresh culture of litmus GFp
Only see the primer => try again with new miniprep
