Background: lambda gt11

Christopher T. Huhta huhta at poe.ccs.itd.umich.edu
Tue Jan 26 13:55:25 EST 1993


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.

The next day I wash the membranes (low stringency: 2x SSC, 0.1% SDS).  I
wash in this fashion:  I open the heat sealed bag with my 'branes in it,
place them (approx. 10) in a 6x9 rubbermaid container with about 500 mL of
2x SSC, 0.1% SDS.  I separate the filters so they don't stick to one
another, and rock them.  I drain the wash fluid and pour another 500 mL
directly back into the dish for the next wash.  I do two 15 min washes at
RT, and two at 50 degrees.

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.  

I might add that I am but a first year med student doing research, and
although I think I have a good grip on my laboratory skills, I don't do
this as a living, and thus my knowledge is somewhat limited.

Thanks in advance!

Chris Huhta
huhta at milton.ccs.itd.umich.edu



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