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From 2012.igem.org

week 12: 16 july - 22 july

Office work

Lab work

Biobrick BBa_J04450 was cloned and miniprepped.

Hepatitis B

Tuesday:

Digestion of

• Hepatitis B PCR product (with pre- and suffix) with Spe1 and Pst1
• BBa_J04500 (an IPTG inducible promoter with RBS) with Xba1 and Pst1
• 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

• Hepatitis B PCR product with BBa_J04500
• the Hepatitis B PCR 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 of the 2 different constructs with DH5α

• 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

out of 20 samples taken from the transformation with the HepB + BBa_J04500 construct – 7 turned out to have the right insert. The colonies transformed with the Hep B + BBa_PSB1K3.ml construct did not have any Hep B inserts

TuYV

• Mach1 electrocompetent E. coli cells were transformed with last week's ligation mixtures. Results were unsatisfying.

• 'Coat Protein + readthrough' gene amplicons were digested, ligated into pSB1K3 backbone and transformed into our electrocompetent Mach1 E. coli cells. After two attempts and a colony PCR, results were still unsatisfying.

PLRV

After a two week quest for PLRV infected plant material, we obtained infected potato leafs from the Dutch General Inspection Service for Agricultural seed and seed potatoes.

The figure below shows the purity of isolated RNA. We isolated RNA of different potato cultivars.

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