I am having problems with my RNA cross-linking to the nylon. I have transferred RNA onto the membrane and the agarose gel is empty after the capillary transfer. The crosslinking was performed at 120 mj/cm2 for 15 minutes. After incubating with prehybridization buffer (5.0x SSC, 0,1% SDS, 3x denhart and MQ), the RNA is lost from the membrane (checked with methylene blue stain onto the membrane). What can be the probable reason?
15 minutes seems like a long time for UV crosslinking.Is this what the manufacturer recommends for this nylon?
I tried 5, 10 and 15 minutes so that the cross-linked RNA remains attached to the membrane but for some reason, it gets removed from the nylon during the pre-hybridization wash.
My feeling is that it depends both on the time and intensity of the UV light. Also UV binding works better on damp nylon not dried nylon but It might be worth trying crosslinking for 30 seconds and 60 seconds rather than 5 mins. A very old but interesting paper is attached. Also dont forget to irradiate the side of the nylon with the rna stuck to it when binding.
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