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Post subject: Hyb temps and washing temp for oligo probes
We have bought the oligonucleotide probes from gene detect. ( GC content of 32% ). I tried them at 37 C , 55 C (hyb temp and washing temperatures respectively ) and I was succesful for the first two times. But I did never get the same results. I used to store them at -20 . How long I should heat the probe and buffer before the hybridisation, at what temp? What hyb, washing temperature is optimum for this probe.
Please help me
Thanks
mini
Tue Apr 28, 2009 5:14 pm
hrn
Rank: Site Admin
Joined: Sep 27, 2004
Posts: 246
Post subject:
Repeated freze-thawing can lower the shelf life of your probe. Did you freeze in aliqots?
Thaw (do not heat) probe and prepare in room temperature.
Using Genedetect hybridization buffer (4XSSC and 50% formamide), optimal hybridization temperature can be calculated using this formula:
T=24.21 + 0.41(%GC) - 500/n
n=length (base pairs)
I suggest hybridization at lower temperature to ensure hybridization, then trying wash temperatures in 5 DegC steps, starting at roomtemp.
Beside protocols, you'll find handeling and storage instructions for Genedetect probes here
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