help needed for oligo screening

ottcr at esvx17.es.dupont.com ottcr at esvx17.es.dupont.com
Tue May 25 16:55:17 EST 1993


In article <1993May21.024038.27297 at nuscc.nus.sg>, mcbzzs at nusunix1.nus.sg (Mr Zhao Zhuoshen) writes:
WROTE:>dear net-friend,
>I am using degenerate oligo(19 mer designed according to peptide
>sequence) to screen cDNA library. The Tm of my oligo is about 42 C. The
>hybridization condition I am using is as following:
>0.5M NaHPO3(ph6.8)
>7% SDS
>10% Formamide
>35 C overnight.
>My problem is that I can not get any signal by using this protocol.
>Has anyone on the net any experience or suggestion about oligo
>screening?  All respones are welcomed.
...
    Here are a couple of tips which should help.
      - Eliminate the formamide completely (at least until your
        protocol is perfected)
      - Cut the SDS to 1%
      - Start with 0.5M NaCl & increase/decrease as necessary
   
    You need to start with a LOW stringency and get a signal
    first, then work on perfecting the system. If using a nylon membrane
    (charged) make sure you hybrdize with a completely clean probe.
   No free labeled nucleotides.
  
   Any further questions - Send E-Mail, I'd be glad to chat.
   Regards, Charly Ott.
  



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