Boston UniversityStatus
From 2007.igem.org
Contents |
What We've Accomplished
Week's (Ambitious) Goals
Week of 6/4:
1. Evaluate the transformation that was done on Friday.
2. Confirm the correct plasmid (pJQ200)
3. Find appropriate restriction enzymes that cut by BLASTing all the plasmid's restriction enzyme sites onto the global transcription factors.
4. Re-design primers for the global transcription factors based on the restriction enzymes we have selected.
5. Order primers.
6. Practice regular (non-error prone) PCR with the primers to check that they work.
7. Incorporate the global transcription factors into the plasmid and transform this plasmid into the E.coli.
8. Conjugate this plasmid into Shewy.
Materials We Need
Error-Prone PCR: Need to Buy
Ligases: Need to Buy?
Short-Term To-Do List
Ordering of Error-Prone PCR Materials: Not completed
Thank-You Letters sent to Pfizer: Not Completed
Thank-You Letters sent to BU ppl: Not completed
Protocols
"Calcium Chloride/Heat Shock Plasmid Transformations"
Question and Answer
Can we get the kanR gene with appropriate sticky ends for BsaI and Tth111I? (DB)
yeah, we can get the DNA template for the kanR gene from the lab. we'll need to design some primers to PCR amplify it out with the BsaI and Tth111I cut sites. after cutting with the restriction enzymes, the kanR will have the appropriate sticky ends. frank suggested that we try ligating the kanR gene to the global regulator gene. (ie. kanR ligated to hlyU) and then stick that into the pJQ200. this way we can select for succesfull hlyU transformations with kanamycin
Ok, good. Do those primers still need to be ordered? And if we follow Frank's suggestion, what will go in place of the gtmR gene?
Also, can someone update the primer design section?
Relevant Publications and Links
http://www.shewybase.bu.edu
https://www.atcc.org/common/catalog/numSearch/numResults.cfm?collection=mb-vector&atccNum=77482
