Team:Paris Bettencourt/Notebook/Trojan Horse/Thursday 1st August.html

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Trojan Horse

1st 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.