Team:Wageningen UR/Journal/week12

From 2012.igem.org

(Difference between revisions)
Line 33: Line 33:
Friday:
Friday:
* gel electrophoresis (1% agarose gel)  of the colony PCR samples
* gel electrophoresis (1% agarose gel)  of the colony PCR samples
 +
[[File:HepB colony PCR 19-7.jpg]]

Revision as of 09:13, 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

HepB colony PCR 19-7.jpg