PCR problem

REGCHAK at USTHK.UST.HK REGCHAK at USTHK.UST.HK
Wed Jul 24 00:31:54 EST 1996


Hi netter,

     I have do a lot of PCR reaction with the SSLPs and CAPs primers to
amplify the Arabidopsis (ler) genomic DNA template, and it work O.K. 
Recently, I have changed my protocol not to include the TaqStart antibody. 
I used the traditional hot start, all the PCR condition are same except no
TaqStart antibody, no PCR products got.  Then I follow the condition
suggested in the " The Plant Journal 1993 written by Ausubel F M and
Konieczny. I increase the no. of amplification cycles and increase time for
polymerization.  Fortunately, I can reproduce a band(in the col background)
by using the ADH primer as before, but not in the ler background.  Before I
start the experiment, I check the template concentration in both
backgrounds, they are the same (about 10-20ng of arabidopsis genomic DNA
20ul PCR reaction).  My genomic DNA were prepared by the CTAB method.

     Could you give me some advice to make the progress, I really want to
reproduce the result without adding the TaqStart Antibody. Any suggestion
will be appreciated.



Regards
Regina






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