User:University of Washington/17 July 2008

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== Lambda Red Recombination of RP4 (Bryan) ==
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Attempted colony PCR of IES010, strain carrying a genomic tetR cassete, following a protocol previously validated by graduate advisor. 
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== RP4 Conjugation ==
== RP4 Conjugation ==

Revision as of 20:08, 18 July 2008

Lambda Red Recombination of RP4 (Bryan)

Attempted colony PCR of IES010, strain carrying a genomic tetR cassete, following a protocol previously validated by graduate advisor.

RP4 Conjugation

Bac-Bac Conjugation protocol #1
·DH5alpha + pCS26-pac
·DH5alpha:RP4 + pCS26-pac

Yeast Shuttle Plasmid

- PEG, LiAc, TE combination plate buffer was made.

- Single stranded carrier DNA was denatured in boiling water for 3 minutes, then placed on ice.

- 1200 uL of yeast in liquid YEPD media was separated into two Eppendorf tubes. These were centrifuged and pelleted, then the supernatant was decanted.

- 5 uL of carrier DNA was pipetted into each tube of pelleted yeast, then 10 uL of pAC88 and 10 uL of sterile water was pipetted into the experimental and control tubes, respectively.

- The mixture was vortexed then centrifuged for 10 seconds to repellet the cells.

- The supernatant was poured off, then 100 uL aliquots of plate buffer was used to resuspend the cells.

- The mixture was vortexed again, then centrigued for another 10 seconds.

- Finally, the supernatant was poured off, and 5 uL of DMOS was added.

- The two mixtures were plated on two leucine-deficient plates then placed in an incubator at 30 degrees Celsius to grow for the weekend.


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