Yeasy PCR screening problem

Yi Yi
Thu Jul 13 13:29:12 EST 1995


Hi there! I can understand how frustrated you are with the PCR problem. I 
have done the yeast PCR screening before without any problems (Does not 
help you, does it?). I think the problem you are having is not due to 
the accessibility of the PCR primers. Often, it is due to other reasons: 
such as primers, solutions, quantity of your templates, dNTPs, etc. It 
could be a long list :). From my experience, I think the problem could be 
due to the amount of your DNA template which is inside yeast. If you use 
haploid cells, you have single copy of your gene on chromosome or few 
copies if on plasmid (I am guessing). You can calculate how many cells 
will give you the amount of DNA template close to 1 ug or 100pmol.  If 
you consider the accessibility of your DNA, You may need more. The 
bottomline is to use more cells in your PCR reaction to generate enough 
PCR fragment to be seen on agarose gel. (Do a titer test)
I hope this may help you. Good Luck!

Yi ZHANG,

>>>No jokes in science<<<

>    Reply 
to: 
Nigel Kenward >    >                          YEASY PCR SCREENING PROBLEM
>                                       
>                                       
>    12 Jul 1995 07:45:00 GMT
>    Dept. Biochemistry, University of Nottingham, U.K.
>   Newsgroups:
>          bionet.molbio.methds-reagnts
>   Reply to newsgroup(s)
>Hi All,
>Any ideas on how to do PCR colony screening on yeast ? I've tried freeze 
>thawing and boiling but that doesn't seem to make the DNA accesible and 
>we don't have French Presses, Hughes Pressure cells etc.
>Any ideas,
>Regards,
>Nigel Kenward
>
>               TEL:+44 (0)115 9249924 ex. 44789
>Dept. of Biochemistry      FAX:+44 (0)115 9422225
>Queens Medical Centre      Email:mbxnjk at unicorn.nott.ac.uk
>Clifton Boulevard           
>Nottingham                                              
>U.K.                   "Back off man, I'm a scientist!"  -- Bill Murray.
--
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@
FAN WANG E-MAIL: FANWANG at GPU.SRV.UALBERTA.CA
@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@



More information about the Methods mailing list