BUGSS:Build-a-BUG:2-4

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Build-a-BUG: Series 2, Session 4

Saturday, March 23, 2013
Noon to 4:00 PM

This is the fourth Build-A-BUG workshop in a series of five on yeast. You will learn more about molecular cloning and continue to hone your basic lab techniques, while building a mating type detector. After troubleshooting an unexpected problem in Session 3, we will actually do ligations (linking DNA) and transformations of competent E. coli cells.

Background reading

• Review Session 1, Session 2, and Session 3
• Functional characterization of the T4 DNA ligase

Protocols

• DNA ligation with T4 DNA Ligase
• One-step preparation of competent Escherichia coli

Materials

• T4 DNA Ligase

Notes

• We ligated all three inserts (MFα2 promoter, BBa_K165057, and BBa_J63002) into the vector (BBa_K801000) using T4 DNA ligase.
• We prepared and transformed competent E. coli with our ligations, then plated on LB+Amp plates. Positive (pUC) and negative (H₂O) transformation controls show the efficiency of the transformation and effectiveness of the selection, respectively.
• Students came in on their own after this session to screen colonies with PCR. They then grew up and isolated promising construct pDNAs.

Whiteboard

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Diagram of desired construct after ligation

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Ligation and transformation protocols

Results

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Plates with transformants after 2 days