BUGSS:Build-a-BUG:2-2

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Build-a-BUG: Series 2, Session 2

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

This is the second Build-A-BUG workshop in a series of five on yeast. You will continue to hone your basic lab techniques, including pipetting and centrifugation, while continuing either your own or somebody else's mating type detector project. This lab will involve restriction endonuclease digests (cutting DNA), agarose gel electrophoresis (visualizing DNA), gel extractions (recovering DNA fragments), and column-based DNA purifications.

Background reading

• Review Session 1
• Structure and function of type II restriction endonucleases

Protocols

• Optimizing Restriction Endonuclease Reactions
• Agarose gel electrophoresis
• QIAquick Gel Extraction Protocol and Handbook

Materials

• EcoRI
• XbaI
• SpeI
• PstI
• 1 kb DNA Ladder

Notes

• We ran samples of our BBa_K801000 (pTUM100) and BBa_K165057 (Kozak + mCherry) plasmid DNA minipreps from Session 1 on an agarose gel with a 1 kb DNA ladder (to measure size). We also ran some BBa_K165057 (Kozak + mCherry) plasmid DNA that had been digested with XbaI and SpeI enzymes.
• We did restriction digests on our BBa_K801000 (pTUM100) plasmid DNA minipreps from Session 1 using EcoRI and PstI enzymes.
• We excised the BBa_K165057 (Kozak + mCherry) DNA fragment from our agarose gels. Then we recovered the DNA using the QIAquick Gel Extraction kit.
• We cleaned up our digested BBa_K801000 (pTUM100) vectors using columns and reagents from the QIAquick Gel Extraction kit.