Hello All. I am looking for advice regarding expression
screening/interactive cloning. I am currently screening a plant
ZapExpress library with a 32P-labeled protein probe. I have been
following a hybridization protocol derived from Current Protocols in
Mol. Bio. which is as follows:
- after lifting, filters are washed for 15 min. at RT in TBS-tween
- filters are then blocked for 4 hrs. at 4 degrees in buffer A (20mM
HEPES, 5mM MgCl2, 1mM KCl) with 5% milk powder
- filters are then hybridized overnight at 4 degrees in approx. 300,000
cpm protein probe/ml of buffer B (20mM HEPES, 7.5mM KCl, 0.1mM EDTA,
2.5mM MgCl2) with 1% milk powder (approx. 40ml for 10x137mm filters)
- before exposing to film filters are washed 3x for 30 min.in 100ml
buffer B.
I am hoping that someone who has successfully performed such a screen
can tell me: 1) should I expect high "background" levels - or, asked
another way, how hot are the filters when you expose to film? My
filters are generally very hot (10-15K on the Geiger counter) and thus
have a high background on film. This background is not noticibly
reduced with different blocks (BSA, 1% milk powder) or overnight blocks
or with increased wash times/volumes.
I am concerned that I may not detect positives over this high
background. Which brings me to my next questions: 2) any suggestions on
reducing this background? and 3) in the primary screen, what should I be
looking for in terms of a putative positive (i.e. intensity, size). I
am used to library screening with a DNA probe . . . will the positives
look the same?
Thanks in advance for any suggestions you can offer!
Vanessa
