Team:Groningen/5 July 2010

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Hydrophobofilm
pushing coatings into a greener future

Week 27, Arend Jan

The pMASK-EH construct is cut with EcoRI and SpeI to get the fragments containing the ChpE and ChpH genes and ligate them in to the expression plasmid. A mix of both genes will be used for ligation and the clones will be checked afterwards for which gene they contain.

- 10ul plasmid pNZ8901-bbs
- 1.5ul buffer Tango
- 0.5ul SpeI
- 3ul MQ
- perform digestion
- add 1.88ul buffer Tango
- add 0.5ul EcoRI

- 17.5ul plasmid pMASK-EH
- 2ul buffer Tango
- 0.5ul SpeI
- perform digestion
- add 2.5ul buffer Tango
- add 0.5ul EcoRI

Mix of fragments containing ChpE and ChpH genes at ~300bp

Restriction of expression backbone with EcoRI and SpeI

Ligation of ChpE and ChpH into pNZ8901-bbs

- 2/5ul backbone
- 14/11ul insert (ChpE+H)
- 2ul T4 buffer
- 2ul T4 ligase

Tried to cut out the insertion from pNZ8901 and replace it with a part from a Plasmid with an identical backbone, all attempts failed however.