skuroda at inherit.biosig.kobe-u.ac.jp
Thu Jun 29 19:42:17 EST 1995
I poosted the same question in this NG several weeks ago. And I tried to modify
the LiAc/PEG method and to introduce the electroporation method. Finally, I got
over 20000cfu/mcg by the modified LiCl/PEG method.
In case of the electroporation method, the efficiency is the same rate as that
of the LiCl/PEG method. But the productivity of transformant cells is lower,
because the cuvette capacity is limited. To get sufficient amount of transformants,
I think 100 cuvettes will be required. In addition, the electroporation method
shows severe strain dependency.
The critical points in LiAc/PEG method are follows:
1) The growth rate:
10% overnight culture seed (OD650=0.2-0.3), 4-5hrs incubation (OD650=0.5-0.6)
2) The cell density/DNA ratio:
To get over 10exp7 colonies, the yeast cells from 1L culture and the 500mcg library
plasmid are required.
3) Carrier DNA (Important):
The sonicated and heat-denatured salmon sperm DNA is required.
The DNA must be denatured completely, which increase the efficiency 100-fold.
The size of the carrier DNA is not so critical, but I think the 5-10kbp average size
The bovine thymus DNA must be avoided, which gives always low efficiency and high
4) DMSO treatment:
It is not so important, but the efficiency incerases about 10-50%.
But ceratin strains show no change after DMSO treatmnent.
5) Heat treatment:
42 degree, 15 min is sufficient. After heat treatment, chilling quickly is important.
6) Recovery process:
After the heat treatment, the incubation of the cells in 1L YPDA for 1hr with shaking
gives good results.
If you have any questions, do not hesitate to ask me.
I hepe you will get good results.
SHUN'ICHI KURODA, PH.D.
Biosignal Research Center
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