Northern blot - (Feb/11/2010 )
I'm doing northern using AMBION kit using the biotin assay
I have few problems:
1. How can I save my membrane wet if in the procedure there is RNA crosslink in 80 degree oven?
2. Tomorrow I need to develop with the BrightStar kit so first do I need to put the X film directly on the membrane or should I put plastic bag ?
3. By the recommendation I'm not sure if I need to expose for 2 hours or 30 minutes
and again if I wait for 24-48hours how to keep the membrane wet?
I need to develop tomorrow so if you can help me soon I'll appreciate it
A bit slow in replying I see...
You can also crosslink with UV, then the membrane won't dry out. Keep it wet in a little buffer (RNase free), or let it dry out, it shouldn't matter too much. I store my membranes dry.
Put your film inside a bag or sandwiched between two sheets of mylar(OHT sheets) otherwise it will stick to the film, and you may accidentally put it through the developer.
Expose for as long as you need to. Start off with 30 min, if that isn't long enough (i.e. you can't see anything on the film at all) try 2 hours. You may need longer or shorter times than either of these.