Background: lambda gt11
Mark S. Whitsitt, N5RJF
msw1633 at sigma.tamu.edu
Wed Jan 27 11:36:00 EST 1993
In article <1k41etINN4kl at controversy.math.lsa.umich.edu>, huhta at poe.ccs.itd.umich.edu (Christopher T. Huhta) writes...
>I have tried doing plaque lifts from a gt11 cDNA library on
>Nitorcellulose filters. I have the problem of VERY high background after
>I wash my membranes. I block with ssDNA (Herring Sperm). I believe that
>I am washing the membranes incorrectly or something. This is my first
>attempt at screening such a library, and there is nobody around my lab who
>has done it before to help me diagnose the problem.
>After 2-3 perhybridization at 42 degrees, P-32 labeled probe is added and
>allowed to hybridize overnight at 42.
I assume from the hybridization temperature that you are using a DNA probe. Is
that correct? Also, what are you using for hybridization buffer? I use the
rapid hyb buffer from Amersham and it works great! I have also used standard
Denhardt's and other made from scratch buffers with similar results. Also, 1
hour of prehybridization at the hybridization temp should be more than
sufficient no matter what buffer you use. I sometimes do a 15 min pre-hyb with
the buffer described in the Stratagene pBluescript exo/mung DNA sequencing
system manual (ie, PE/formamide)
>The next day I wash the membranes (low stringency: 2x SSC, 0.1% SDS). I
>wash in this fashion: ... 500 mL of 2x SSC, 0.1% SDS... I do two 15 min
>washes at RT, and two at 50 degrees.
I would recommend 2 15 min washes at RT with 2x SSC, and 0.5% SDS and then 2
15-30 min washes at 50 degrees with 0.2x SSC/ 0.5% SDS. This has always worked
for me whether doing northerns or plaque lifts or such. The only changes are the
temperatures for the last 2 washes. Usually I wash at 60-65 degrees for a
northern. You'll have to play with it to get it just right. If you increase
the temp you will reduce the background, but you may make the wash too stringent
if you are not careful.
>Changing SDS and SSC conc. doesn't change anything. I must be doing
>something wrong. My membranes (after autoradiography) come out with some
>clear ares (shoing some hybridization where appropriate) and the rest of
>the membrane is black as night.
>huhta at milton.ccs.itd.umich.edu
Mark S. Whitsitt, N5RJF Texas A&M University, Dept of Biochemistry
Internet: msw1633 at summa.tamu.edu College Station, Tx. 77843-2128
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