No replies to this topic

[scientist]

The gene I am interested in has 3 isoforms that we wish to clone the cDNAs.

Isoform 1 has the same sequence as isoform 2, but isoform 2 has an insert in the middle making the cDNA about 200 bases longer. We are using primers to amplify isoform 2 and they also amplify isoform 1 (there is nothing unique with isoform 1). When we do a RT-PCR, we get two bands on the gel of the correct sizes corresponding to both isoforms. However if we isolate isoform 2 from the gel and re-PCR for cloning it amplifys as the size of isoform 1.

We have also tried direct cloning of the RT-PCR product and we only see isoform 1 in the clones.

Any advice on how to clone isoform 2 would be greatly appreciated.