Northern blot exposure time - (Apr/01/2007 )

I'm new to Northern blotting. How do you determine your exposure time after probing/washing the blot?

We use autoradiography, I expose at -80 with an intensifying screen.

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It depends on how hot your probe is, for a start. Quickest way is to do a short exposure, say, 5 minute, and see what comes up. If you get nothing, try a couple of hours, and check again.

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Optimal exposure times must be empirically determined, but the following intervals are offered as a guide. In these cases, radioactive decay was measured on a Ludlum model 3 survey meter with a 44-3 detector, where 1 mR/hour is approximately 2500 counts per minute.

For genomic DNA, cpm are usually at background. Expose a minimum of 4 days.
For cloned DNA, use the following chart to estimate exposure times:

CPM just above membrane Exposure Time
< 0.2 K Overnight
0.5 K 3-5 hours
1 K 30 minutes
3 K 20 minutes

(http://omrf.ouhsc.edu/~frank/AUTORAD.html)

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Thanks! That helps.

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