help for oligo hybridization

[Timothy Bushnell]

In Article <38nigg$2u3 at nntp.Stanford.EDU>, minmin at leland.Stanford.EDU (Limin
Li) wrote:
>I have been trying different conditions for oligo(25-40 mers) hybridization.It
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>seems I always get strong background or nothing(of course I tried different
tem\
>peratures and washing conditions). I am using Amersham's filter. I checked the
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>oligos on computer, they all ok.
>Any suggestions would be greatly appreciated.
>Thanks in advance.

    I have no personal experience with the Amersham filters.  For our
hybridizations, we typically use the membranes from S&S. We have
successfully used them with all sorts of probes, especically oligos.  

    What type of blots are you doing?  There could be several problems,
ranging from insufficient blocking, to high washes, to powder from golves
getting on the membrane (causes background).

    How are you calculating TH?  Try the equation:

    TH= (2C(#A:T bp) + 4C (#G:C bp)) - 5    

    I hope that this helps.
Timothy Bushnell
Plant research group

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