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Contents

What We've Accomplished

Plasmid Choice

Restriction Enzyme Choice

Primer Design

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

Restriction Enzymes: bought

Ligases: Need to Buy?

TOPO kit: available in lab

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)

Relevant Publications and Links

http://www.shewybase.bu.edu

https://www.atcc.org/common/catalog/numSearch/numResults.cfm?collection=mb-vector&atccNum=77482

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