3' RACE creates too big band doesn't leave well - (Apr/11/2013 )
I ran the 3' RACE a couple of times and each time there is a visible band that is too large to leave the well. Ran it with a 10,000 bp ladder and the ladder reached middle of gel before I stopped. Did the 2% gel like the protocol calls for and a 1.5% gel. Also verified my primers are cutting by running with other primers and the band wasn't there. I am looking for a band around 3kb.
A big band implies that you have some genomic DNA contamination or that there is significant secondary structure in the product that you are producing. for 3000 bp (3kbp) I would run a 1% gel.