help! plasmid reisoltion/two hybrid

Rich Buckholz RGB12955 at glaxo.com
Thu Mar 14 16:34:53 EST 1996


Try this.  I got it from this group some time ago and made a few
modifications.  It works every, every, every, every, every, every, every,
every, time!

Also, keep some 3AT around all the time (use -his media) as some -AD
fusions are poorly tolerated.

Extraction of Autonomous Plasmids from Yeast
(see alternate protocol later)

1. Grow 2 ml of culture under selection to saturation.

2. Transfer 1.5 ml to microfuge tube, spin top speed, 30 seconds, to
pellet.  Aspirate supernatant.

3. Resuspend pellet in 200 ul zymolyase solution made freshly from stocks 
(see recipe below).  Pellet may be vortexed to resuspend.

4. Incubate 37C, 2 hours.

5. Add 200 ul Promega Wizard miniprep solution 2 (cell lysis
solution--this is nothing magical; just alkaline lysis solution).  Mix
well by inversion.  The mixture normally appears clear or may have a glob
of lysed cells + chromosomal DNA.

6. Optional:  incubate at 65C, 5 minutes, to aid lysis.

7. Add 200 ul Promega Wizard miniprep solution 3 (neutralization
solution).  Mix gently by inversion.

8. Optional:  chill on ice 15 minutes.  This is probably more important if
you heated 65C, 5 minutes (step 6).

9. Microfuge top speed, 5 minutes.

10.   Transfer the clear supernatant to a fresh 1.5 ml tube containing 1
ml of the Promega Wizard miniprep DNA purification resin.  Mix for 20
seconds by inversion, incubate RT at least 1 minute.

11.   Subsequent steps for column loading and washing under vacuum or by
using a syringe are as in the Promega Wizard miniprep protocol.

12.   Elute DNA from the resin with 50 ul sterile distilled water.  For
transformation into CaCl2-competent bacteria, use 5-10 ul of the DNA.  For
2u yeast plasmids and highly competent bacteria, 1 ul may be sufficient.

Note:  this protocol calls for the Promega Wizard miniprep DNA
purification kit.  It will probably work with any alkaline lysis-based
miniprep protocol.


Zymolyase solution:


stock         volume per 1 ml solution      final                             

2 M sorbitol         600 ul                    1.2 M
1 M KPO4 pH7.5     100 ul                   0.1 M
4 mg/ml zymolyase 100T* 100 ul             400 ug/ml
H2O                   200 ul  

*if using zymolyase of different specific activity, adjust stock
concentration or volume accordingly.  Zymolyase stock may be made ahead of
time and stored as aliquots at -20C.  It is a tough enzyme that may be
frozen and thawed repeatedly; it may require vortexing to dissolve. 
Occasionally the powdered enzyme has an excipient that dissolves poorly;
in this case vortex and use as a suspension.  Zymolyase is available from
Seikagaku (no, I didn't make that up), ICN or Miles Biochemicals.  If
anyone finds a more widely available source, please let your colleagues
know.

Alternate Protocol:
Extraction of Autonomous Plasmids from Yeast

1. Pick single isolated colony with toothpick into 100 ul zymolyase
solution, made freshly from stocks, in 1.5ml microfuge tube.  Cells may be
vortexed to resuspend.

2. Incubate 37C, „1 hour.

3. Add 100 ul Promega Wizard miniprep solution 2 (cell lysis
solution--this is nothing magical; just alkaline lysis solution).  Mix
well by inversion.  The mixture normally appears clear or may have a glob
of lysed cells + chromosomal DNA.

4. Incubate at 65C, 5 minutes, to aid lysis.

5. Add 100 ul Promega Wizard miniprep solution 3 (neutralization
solution).  Mix gently by inversion.

6. Chill on ice „5 minutes.

7. Microfuge top speed, 5 minutes.

8. Pour the clear supernatant into a RC-10ml Rainin pipette tip,
containing 0.5 ml of the Promega Wizard miniprep DNA purification resin,
mounted on a vacuum manifold.  Incubate RT at least 1 minute.

9. Subsequent steps for column aspiration and washing under vacuum are as
in the Promega Wizard miniprep protocol.

10.   Elute DNA from the resin with 30 ul sterile distilled water 70°C,
„1minute.  Transform 10 ul into 100ul subcloning efficiency DH5a-competent
bacteria.

Note:  this protocol calls for the Promega Wizard miniprep DNA
purification kit.  It will probably work with any alkaline lysis-based
miniprep protocol.




In article <v01540a01ad6ca2c47e9c@[129.132.49.227]>,
froemel at cell.biol.ethz.ch (Christine Froemel) wrote:

> Hi there,
> as I'm really new in yeast, I got quite a basic problem with the two hybrid
> system or better with my transformants. The amount of colonies I get I'm
> quite content with, but I'm hardly able to reisolate any of the transformed
> plasmids out of these positive colonies.
> I use two strains: Y153 and HF7c (Clontech) together with the plasmids pAS2
> and pGAD424. I transformed following the protocoll from clontech
> (LiAc/ssDNA/PEG). (no record suspicious number of transformants, but
> acceptable.)
> To reisolate plasmids I tried two protocols: A.C.Ward (Nucl.AcidRes.Vol.18,
> No.17, 5319) and R.Soni, J.Murray (Nucl.Acid Res. Vol.20, No21, 5852) as
> well as a protocoll for direct PCR (G.M.Sathe et al. Nucl.Acid Res.Vol.19,
> No.17, 4475) - all of them with really scanty or no results.
> Both strains aren't leaky with LEU and TRP (the selection markers on the
> two plasmids); I checked my media, retried with freshly prepared stuff...
> It really should work!
> Thanks a lot in advance for any advice!
> Christine
> froemel at cell.biol.ethz.ch



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