Team:Wageningen UR/Journal/week12

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== Lab work ==
 +
 
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''' Bricking Hepatitis B '''
 +
 
 +
Tuesday:
 +
 
 +
* Digestion:
 +
of the Hepatitis B PCR product (with pre- and suffix) with Spe1 and Pst1
 +
of BBa_J04500 (an IPTG inducible promoter with RBS) with Xba1 and Pst1
 +
of BBa_PSB1K3.ml (linearized plasmid backbone) with EcoR1 and Pst1
 +
* PCR purification on the digest as a replacement for the heat inactivation
 +
 
 +
Wednesday:
 +
* Ligation:
 +
of the Hepatitis B PCR product with BBa_J04500
 +
of the Hepatitis B product with BBa_PSB1K3.ml
 +
The ligations where done in duplo – once following the standard iGEM protocol and once using equimolar amounts of Hep B and the vector (DNA concentrations where measured by using NanoDrop)
 +
* Electrotransformation in E.coli
 +
* Grow transformed E.coli on plates containing kanamycin
 +
A plasmid with a GFP coding device was used as a positive control (growing on a plate containing ampicillin)
 +
 
 +
 
 +
Thursday:
 +
* Colony PCR
 +
of 20 colonies transformed with the Hep B + BBa_J04500 construct
 +
of 10 colonies transformed with the Hep B + BBa_PSB1K3.ml construct
 +
 
 +
Friday:
 +
* gel electrophoresis (1% agarose gel)  of the colony PCR samples

Revision as of 09:01, 31 July 2012

Lab work

Bricking Hepatitis B

Tuesday:

  • Digestion:

of the Hepatitis B PCR product (with pre- and suffix) with Spe1 and Pst1 of BBa_J04500 (an IPTG inducible promoter with RBS) with Xba1 and Pst1 of BBa_PSB1K3.ml (linearized plasmid backbone) with EcoR1 and Pst1

  • PCR purification on the digest as a replacement for the heat inactivation

Wednesday:

  • Ligation:

of the Hepatitis B PCR product with BBa_J04500 of the Hepatitis B product with BBa_PSB1K3.ml The ligations where done in duplo – once following the standard iGEM protocol and once using equimolar amounts of Hep B and the vector (DNA concentrations where measured by using NanoDrop)

  • Electrotransformation in E.coli
  • Grow transformed E.coli on plates containing kanamycin

A plasmid with a GFP coding device was used as a positive control (growing on a plate containing ampicillin)


Thursday:

  • Colony PCR

of 20 colonies transformed with the Hep B + BBa_J04500 construct of 10 colonies transformed with the Hep B + BBa_PSB1K3.ml construct

Friday:

  • gel electrophoresis (1% agarose gel) of the colony PCR samples
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