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.
