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plasmid rescue tips and tricks

Fatima Cvrckova FATIMA at AIMP.UNA.AC.AT
Mon Mar 14 05:31:03 EST 1994


Dear Margaret et al.,

What follows is the yeast DNA prep protocol our lab used for both Southerns and 
plasmid rescue, before we switched to the boiling prep. It is tedious but it
usually worked.

SOLUTIONS
SCE:          1M-sorbitol
              0.1M-Na Citrate
            0.06M-EDTA (autoclaved)
               pH7.0

SCE/zymolase/2-ME mix:  to each ml of SCE :
                                1-2mg zymolase 5000 and 8µl 2-ME 

SDS solution:          2% SDS
                               0.1M-Tris/Cl pH9.0
                               0.05M-EDTA

5M-NH4OAc (filter sterilised)

5M-KOAc (autoclaved?)

TE

(NOTE: "GDW" means "glass distilled water" means "you can use MonoQ water")

Start with either 10ml of -leu, -trp, -ura
                       or   5ml of YEPD  stationary phase cells.

Spin cells down 2-3min in benchtop 2-3000rpm or leave o/night in cold room to settle 
(in which case pour off medium carefully).

Add 1ml GDW to cell pellet .

Resuspend by vortexing and pour into a 1.5ml microfuge tube.

Spin 20sec microfuge, pour off GDW.

Resuspend cells in 0.2ml of SCE/zymolase/2-ME mix.  Incubate 37ºC with occasional 
shaking for 30-60min.

Spheroplasting can be checked by adding an aliquot (1µl) of cells to a drop of 1% 
SDS solution and viewing with microscope.  Intact cells are bright whereas 
spheroplasts are dark and ghostlike.

When complete, add 0.2ml   of SDS solution.  Mix and vortex briefly.

Heat at 65ºC for 5min.

Add 0.2ml 5M-KOAc.  Again mix by vortexing (briefly).

Leave at 0ºC for 20min.

Spin at high speed for 5min in either an angle or horizontal grey rack microfuge.  
Transfer 0.4-0.5ml supernatant using a cut off P1000 tip to a fresh tube.

Add  0.2ml 5M-NH4OAc
        1.0ml isopropanol
Mix well and spin at low speed for 15-30sec in an angle microfuge.  NOTE.  You do 
not want a compact pellet.

Spin again at low speed, and pour off supernatant, remove remaining sup by 
aspiration.

Dissolve in 90µl TE.
When dissolved, add 10µl 5M-NH4OAc, mix and then add 200µl isopropanol.  Mix well by 
gentle shaking.  A fibrous ppt of DNA will form.   Let it sink to the bottom of the 
tube- where it should stick.

If you do not have such a precipitate, centrifuge at low speed for 30sec instead.

In either case, pour off supernatant carefully.
Rinse pellets 1x in 80% EtOH, remove dregs of 80% EtOH by a low speed spin followed 
by aspiration and dissolve pellets in 50µl of TE.  Store at -20ºC.

For plasmid rescue, don't use more than 1 µl for transformation.

****

However, I suspect that the DNA prep method is NOT the reason why you can't
rescue the plasmid (boiling prep works fine for me). Some yeast DNA preps
inhibit transformation; try to transform diluted DNA! Quite often problems
are due to competent E. coli cells; make sure that your cells are really
competent, and if you can, electroporate.

Good luck!

Fatima Cvrckova (fatima at aimp.una.ac.at)



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