Thanks jerryshelly1 for your reply. I went ahead and ran qPCR with a 15 sec elongation time, since the amplicons were less than 250 bps (if 1 min = 1000 bp then 30 sec = 500 bp and 15 sec = 250 bp) then confirmed the quality of the data by running the qPCR products on a 2% agarose gel. I believe the bands that are present are of good enough quality for a proper qPCR. Thanks for your help again.
I've just been putting the 0.1% gelatin (autoclaved) coated dish in the incubator (37C) for 30 min, aspirate, then use. Results are fine so far. You let your surface dry before seeding?
To address your question about the gelatin not firming up, I'm not sure why that is the case. Perhaps an enlightened one will shine their light upon this. Only my 2% gelatin firms up at 4C.
Thanks guys, these are very good points. I am in the process of going back over the data and throwing out anything that was meant to be mRNA data that was used for the primer above (primers on intron). I really do appreciate the help!
Interesting! The purpose is for PCR, we've been using for awhile with good results. The guy who made them is no longer with us. How did you find that it binds to the 2nd intron?
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