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Team:Wageningen UR/Journal/week12
From 2012.igem.org
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'''TuYV''' | '''TuYV''' | ||
| - | * Mach1 electrocompetent ''E. coli'' cells were transformed with last week's ligation mixtures. | + | * 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. | * '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. | ||
Revision as of 12:30, 18 August 2012
week 12: 16 july - 20 july
Office work
Lab work
Biobrick BBa_J04450 was cloned and miniprepped.
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
colony PCR of 20 colonies transformed with the Hep B + BBa_J04500:
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.
