cracking?

Rogier rogierNOroSPAM at bio.vu.nl.invalid
Thu Jul 20 13:40:21 EST 2000


Hiya,
Here's two a cracking methods that always worked for us. Note
that the cracking buffers for the two protocols are NOT the same.

cracking cells from a liquid culture:
- spin down 1 ml of cells
- add 30 ul cracking buffer
- resuspend all the cells
- 15 min. at room temperature
- spin down debris for 5 min. (not longer)
- put plasmid on gel

cracking buffer is
50 mM Tris pH 6.8 (same pH as Tris for an SDS-PAGE stacking gel)
2 mM EDTA
0.4 M sucrose
1 % SDS
0.01 % bromophenolblue
1 ug/ml RNase A

If you don't want to grow a culture, crack the clones straight
from the plate:
- put clone in 50 ul 10 mM EDTA pH 8
- add 50 ul (freshly made) 2x cracking buffer
- vortex till all cells are suspended
- 5 min at 70 C, then cool to room temperature
- add 1.5 ul 4M KCl and 0.5 ul 0.4 % bromophenolblue
- vortex
- 5 min on ice
- spin 3 min at 4 C, top speed
- run 25 to 50 ul of the sup on a 0.7 % agarose gel (make big
slots! you can make slots bigger by putting tape on the comb)

2x cracking buffer is
2 ml 5M NaOH
2.5 ml 10 % SDS
10 g sucrose
water to 50 ml

Cya,
Rogier Stuger
rogier AT bio.vu.nl


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