Probing lambda libraries with oligonucleotides
rogera at atcg.com
Fri May 12 11:33:05 EST 1995
>Steven Goldberg <goldberg at bms.com> writes:
>>1. Transferring and fixing phage DNA: Some procedures say immerse
>>the filters in the denaturation and neutralization; others claim this
>>gives diffuse signals.
Having done this procedure many many times I find that for Lambda it did
not make a difference.
>>2. End-labeling oligonucleotides: How much DNA is needed, how much
>>label (again, I've seen anywhere from 5 t0 25 ul of gamma-dATP rec-
>>commended). How about the ECL 3' labeling kit or similar?
I labelled 100 ng of probe and I used 40 - 60 uL of gamma-ATP. I thought
it seemed like a lot at first but remember this drives the reaction. If
you are thinking of costs, the cost of repeating the experiment is much
more. Beside 1000 uCi is about the same price as 500 from some suppliers.
>>3. Pre-hybridization and hybridization conditions. Some use SSPE,
>>others SSC; some include pyrophosphate; others leave out Denhardt's
>>in the hybridization.
SSPE is much better buffer but it works either way. Calculate the TM and
subtract 15 degrees. In fact if you do the washes right you can go a bit
lower. Not too low you need to have hybridization.
>>4. Wash conditions.
USE Tetramethylammoniumchloride. This allows you to set VERY specifically
the degree of divergence allowed. I screened several libraries using a 28
mer and allowed for only 2 - 3 bases mismatch. It worked perfectly every
time. In fact I even used a 21 mer with the same result.
I published a paper showing the number of repeat copies in a sea urchin
genome using this technique
Anderson et al (1994) Development Biology 163, 11-18
If you have any questions feel free to write and ask.
>>Thanks for any suggestions.
Anderson Publications PO Box 36
Info at atcg.com Monrovia, CA 91017-0036
URL http://www.atcg.com/atcg/ Voice/FAX (818) 358-0579
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