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Team:Groningen/5 July 2010
From 2010.igem.org
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[[Image:08-07-10gn.jpg|200px|thumb|left|Mix of fragments containing ChpE and ChpH genes at ~300bp]] [[Image:08-07-10-2gn.jpg|200px|thumb|left|Restriction of expression backbone with EcoRI and SpeI]] | [[Image:08-07-10gn.jpg|200px|thumb|left|Mix of fragments containing ChpE and ChpH genes at ~300bp]] [[Image:08-07-10-2gn.jpg|200px|thumb|left|Restriction of expression backbone with EcoRI and SpeI]] | ||
<br style="clear: both" /> | <br style="clear: both" /> | ||
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| + | Ligation of ChpE and ChpH into pNZ8901-bbs | ||
| + | <pre>- 2/5ul backbone | ||
| + | - 14/11ul insert (ChpE+H) | ||
| + | - 2ul T4 buffer | ||
| + | - 2ul T4 ligase</pre> | ||
Revision as of 15:31, 23 October 2010
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
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.
