Tuesday, November 18, 2003

Description
Colony Plasmid Rescue

Procedure
Start with a fresh plate of yeast, with large colonies grown for 3-4 days.


Prepare eppendorfs with 20 l aqueous buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0).


Pick colonies from plate with a pipette tip and resuspend in the aqueous buffer.


Add 10 l phenol and 10 l chloroform (or 20 l phenol:chloroform) to each eppendorf tube.


Add 1/3 eppendorf cap (0.65 ml tube) of glass beads.


Vortex 5 min. at speed of 4-5 in multihead vortexer at room temperature.


Spin for 5 min. at maximum speed in a microcentrifuge.


Thaw transformation competent E. coli cells on ice.


In test tubes (for microfuge tubes see below; otherwise use 14-ml snap-cap tube) on ice add:

a) 1 l aqueous phase (if you experience low yield, try using more)

b) 120 l competent cells


Mix and incubate on ice for 30 min.


Heat shock for 90 sec at 42 deg C, then immediately add 2 ml of NZY broth to each test tube. (LB will also work, though richer media is usually better)


Shake gently at 37 deg C for 1 hr. (If using microfuge tubes, rotate at 37C in roller drum)


Pellet cells for 5 min. at 3 krpm in table top centrifuge.


Pour off the supernatant, resuspend the pellet in the residual liquid, and plate the entire suspension on selective medium.


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