Backgound in Northern - (Oct/22/2008 )

Hi everyone,
I am starting doing Northern with radioactive probes(32P dCTP) made by PCR. So far(after more than a month), what I can only get is very high background(strong radioactivity all over the membrane) . PCR works with cold dCTP, but I am not sure my probe is correctly made by PCR because I use much less HOT dCTP in the PCR reaction and i can not see any band in agrose gel. And I am also thinking that the Nylon or the washing buffer is wrong. So, please give me any suggestion where should I work with?

*PCR: final conc.: cold dA/G/TTP 200uM; hot dCTP 0.6uM(300 fold less that dATP)
*Probe is purified by quick spin colomn before adding to the hybridization mix
*Membrane: Nytran SPC 0.45 Nylon tansfer membrane
*Pre-hybridyzation: 30% Formidhyde, 5X SSC, 0.2% SDS, 100ug/ml ssDNA selmon sperm, 20ug/ml polyA RNA, 20 mM Tris---@42 C
*Washing: 2X SSC, 0.1%SDS, 0.025M NaH2PO4----@65 C
0.1X SSC, 0.1%SDS, 0.025M NaH2PO4-----@42 C

Any suggestion will be strongly appreciated!!!

Dan

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do you purify the radioactive pcr product from the reactants? if not then you are introducing radioactive dctp to the membrane along with the pcr product. that could account for the high background.

do you confirm the radioactive product before you use it? with less dctp you may end up with less product. you may want to consider adding cold dctp to equalize the concentration with the other dntps. the product won't be as hot but should be hot enough.

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Thank you, I use quick spin column after PCR to purify the radioactive product. Do I need to gel purify it? And for my calculation the hot dCTP is 300 fold less than other dnTPs, that is a lot. So to compensate, how much cold dCTP should I add? How about 10 fold of hot dCTP?
You don't think the membrane and washing system are the problem, and I should focus on the probe making right?

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i would equalize the dntps, but i'm not too smart. 10x may be okay. either way, the probe won't be as hot.

oops, i didn't notice that you cleaned the pcr product before i commented (as i said, i'm not too smart), just make sure you don't overload the spin column or you will get the reactants coming out with the product.

are you hybridizing too long? you don't mention.

washes are fine, you may try repeating or even increasing the temperature (you can also rewash after exposing, if you find the background too high).

maybe prehybridization can be increased (time or dna or both).

nytran is what we used. so it's okay.

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