User:University of Washington/17 July 2008

From 2008.igem.org

Revision as of 21:56, 17 July 2008 by Alecn (Talk | contribs)

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.


Back to Team:University_of_Washington/Notebook#Notebook