I have started to do 5' RACE after getting the 3' end of the gene as I have already mentioned in my other thread. I have designed 4 reverse primers to do that. After cDNA synthesis (I did it at 42 and 55 C) I have ran them on Agarose gel. my expected band size is 1.4kb. From agarose gel I have cut the gel from 1 kb to 2 kb portion to avoid unwanted cDNA s. Then I have purified it with Qiagen kit and then tailed them with dATP and TDT. then I have diluted the samples 50 times and did PCR with other GSp s and OligodT-Anchor at 50 and 58 C by Taq. But getting 100-200 bp band like heazy things. I have already checked my GSP s and found that they are absolutely ok and working fine at these temp. (I did PCR with them by using my 3' RACE cloned product and M 13 primers)I have repeated them several times but got the same results. Please help me out of this. Thanks in advance.
Tanks and regards