24, 96 well plasmid rescue

Steve Kron kron at WI.MIT.EDU
Thu Apr 6 18:04:11 EST 1995



Hi Friends ,

Assuming there is anyone interested, like you two hybrid types, here are 24
well and 96 well DNA preps for plasmid rescue:

(See Manuel Claros' "plasmid DNA miniprep" from Jan 21, 1994 for protocol
that I only slightly modified. Thank you Manuel!)

The following is still under development (!) but seems to work fine for the
first few hundred tried.  Probably can shorten a few steps, etc.  I
recommend you use a multi-pipettor like the Eppendorf.

In 24 well plate
1) Grow 2 ml overnight culture of picks in 2x synthetic media with 4%
dextrose
2) Spin out cells in refrigerated centrifuge with plate holders.  (If you
don't have one, you're pretty much out of luck.) 
3) Shake off sup
4) Resuspend in 1 ml H20
5) Spin, shake off sup
6) Resuspend in 1 ml lysis buffer (50 mM Tris 7.5, 10 mM EDTA, 0.3% BME)
7) Spin, shake off sup
8) Resus in remaining liquid
9) Add 25 ul 5 mg/ml Zymo 100T in lysis buffer. (OK if not all in
solution.)
10) Incubate on shaker 1 h at 37 C.  (Lysis should be obvious.)
11) Add 25 ul 10% SDS.  Lysate will clear.
12) Mix, let sit 1 min
13) Add 100 ul 7.5 M NH4OAc.  (Massive precipitate.)
14) Mix gently
15) Freeze -80C, 15 min
16) Spin 15 min. (Clear sup.)
17) Transfer sup to new plate
18) Add 75 ul isopropyl OH
19) Mix, spin, shake off sup
20) Add 1 ml 70% EtOH, mix, spin, shake off sup
21) Air dry, resus in 100 ul TE

Transform about 2-5 ul into Hanahans, etc.

For 96 well, adjust growth volume down to 300 ul and you can use an 8 well
pipettor at all steps.  

Good luck.  





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