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for your kind help about yeast transformation again

for your kind help about yeast transformation again - Protocols and Methods Forum

for your kind help about yeast transformation again - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 09-01-2003, 01:32 AM
shude yang
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Default for your kind help about yeast transformation again



Hello:

I need you kind help urgently. I meet a great
difficulty in yeast transformation and plasmid
exctration.
I constructed a cDNA library of a grass-cynodon
dactylon using a E.coli-yeast shuttle vector-pFL61 at
first.Then I used a whole library to transform a
Arsenic(As) sensisive yeast,FD236-6A(MATa ,ura3-52,
trp1 63, leu2 1, his3 200, acr3- 1::URA3 GAL2), I
screened transformed yeast clones on trp-dropout
medium firstly, afterward I screened As resistent
clones on YPD medium with a high level of As(400um).I
have got some positive clones,but i can not isolated
any plasmid. The worst is that, I can not know where
the errors occoured.
I found that the FD236-6A can also grew on
trp-dropout medium if they were cultured for over 3
days,
even though they grew poorly.What's more, if I put
more As sentive yeast cells-FD236-6A on the
As-including plate(for example, if i pick a small
yeast clone from YPD medium plate),I found that they
can grew well.I also found that transformation
procedure (I refered to LiAc method)could also improve
the resistance ability to As of FD236-6A.What is
happened? I can not know how can i screen postive
clones(that is to say,As resistent clones) from
transformed yeast. Please give me your kind help.Thank
you.

sdyang


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