Team 2 Notes: Difference between revisions
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#Map- we want to look at where the cassette was inserted. | #Map- we want to look at where the cassette was inserted. | ||
##Need to know sequence of DNA that the protein binds to | ##Need to know sequence of DNA that the protein binds to | ||
[[Template:SBB10Project-11097 | Zinc finger domains project page]] for reference |
Revision as of 14:35, 29 March 2010
3/29/10: Protocol planning
Assumed starting materials:
- cells transformed to contain composite part w/ genR flanked by terminal repeats, self lysis device, and prepro-transposase under conditional origin of replication (R6K)
- buffer containing pUC-ampR plasmid (how much?) and tetracycline (how much?)
Steps:
- Add cells to buffer and let sit for ??? min. Add heat? Agitate? Incubate? (cell lysis, transposase reaction)
- Find self lysis protocol and transposase info (heat required, how long to react)
- Lysate buffer (cell junk, desired plasmid, old genR plasmid, chromosomal DNA, transposase) needs to be mini-prepped. (standard protocol) (something special for tetracyclin?)
- Now we have desired plasmid and old genR plasmid in water. Use to transform cells (basic transformation protocol). (Don’t transform into Pir cells!!!)
- Plate transformed cells on amp/gen plates.
- Select colonies.
- Map- we want to look at where the cassette was inserted.
- Need to know sequence of DNA that the protein binds to
Zinc finger domains project page for reference