lysis of YAC containing yeasts help

Anne Forus aforus at ulrik.uio.no
Mon Apr 26 04:04:38 EST 1993


In article <1993Apr19.083139.25296 at gserv1.dl.ac.uk>,
IBELGAUFTS at vms.biochem.mpg.de wrote:
> 
> Has anyone a protocol for isolating DNA for PFGE from YAC containing yeast cells?? We have tried various protocols but they do not seem to work properly.

We use two different protocols, both seems to work very well. The first one
is somewhat quicker than the second, but the outcome is equally good.

1.st PROTOCOL

* Prepare a solution of 2% Low Melting Point agarose in 1 x SCE
Add beta-mercaptoethanol to final conc. of 30 mM
1 x SCE: 1M sorbitol, 0.1 M Sodium Citrate pH 5.8, 0.01 M EDTA pH 7.5

* Grow yeast cells o/n (or longer) in a selective medium, 100 ml cultures:
AHC URA- (1l):
1.7g yeast nitrogene base w/o amino acids and (NH4)2 SO4 (DIFCO)
5g of (NH4)2 SO4
10 g Casamino Acids (DIFCO - Vitamine Assay quality)
20 G Glucose
20mG Adenine Hemi Sulphate (SIGMA)
20mG L-Tryptophane 

pH should end up at 5.8 or slightly higher

* Pellet 50 ml of cell culture (10 min 500 xG), resuspend the cells in cold
50mM EDTA

* Count the cells, pellet again and resuspend to a final concentration of 2
X 10E9 cells/ml in spheroblasting solution:

1x SPH : 1 M D-Sorbitol
         20mM EDTA
         14mM beta-mercapto ethanol
          20 units/ml Lyticase

Prewarm at 37 deg. C.
Add an equal volume of the 2% LMP agarose in 1x SCE
mix and dispensem the solution into slots of plug mould

* Let solidify on ice and extrude plugs into a 50ml tube filled with

1M Sorbitol
20mM EDTA
14mM beta-mercapto EtOH
10mM TRIC-Cl pH 7.5
20 U /ml Lyticase

Incubate 2 hrs at 37 deg.C

* Cool on ice 5-10 min
*Replace with Yeast lysis solution YLS:
YLS:
1 % Lithium Dodecyl Sulphate
100 mM EDTA
10mM Tris-Cl

1 hour at 40 (37-45) deg. C

* Cool again, replace YLS with fresh YLS and incubate o/n as previously

* Replace YLS with Fresh YLS

PLUGS should now be stored at room temperature. Before use, remove the LDS
by dialysis several times in TE pH 8 (2 x 30 min, 50 deg. C, 2 x 30 min
r.t)
 
2.nd PROTOCOL (a little more time consuming):

* Grow Yeast cells as described above (48 hrs prefered)
* Harvest by pelleting 5 min at 5000 rpm. Resuspend in 1/5 of original
culture volume of 0.05M EDTA pH 8.0 and pellet again

* Resuspend in 0.05M EDTA and count the cells
* Resuspend to the desired concentration 
* Add 25 micro l of 2mg/ml Zymolyase 100T pr ml of cell suspension, mix
briefly and warm to 37 deg.C
* Add an equal volume of 2%LMP agarose in 0.125M EDTA
and make plugs as described above
* Place the plugs in 0.5M EDTA pH 8 and 7% beta-mercapto EtOH
Spheroblasting o/n at 37 deg.C
* Discard the solution and rinse 3 x in 50mM EDTA
* Place the samples in 0.5M EDTA pH 8.0, 1% Sarcosyl and 0.5 mg/ml protease
K
Incubate at 50 deg. C for 24 hours, gently shaking
* Wash several times with 0.5M EDTA and store the samples in the same
solution

The samples should be dialysed in TE before using restriction enzymes

This second procedure is essentially as described by Dr. Birren at the CSH
course "Cloning and analysis of large DNA molecules", 1992

Good luck.


Anne Forus                 aforus at ulrik.uio.no
Dept for Tumor Biology
Inst for Cancer Res
The Norwegian Radium Hospital
Montebello
N-0310 OSLO Norway



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