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User:University of Washington/17 July 2008
From 2008.igem.org
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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