Team:Paris Bettencourt/Notebook/Trojan Horse/Wednesday 14th August.html

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Trojan Horse

14th August


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