Hi! This is my first time here, and I really need some advice.
I've found a gene to be alternatively spliced. There are three isoforms: the full length (2.5kb), splv1 (0.4 kb sorter) and splv2 (0.2kb shorter). In splv1 and splv2, 400bp and 200bp are cut out of the middle of the gene, respectively.
I have the full length cloned into a plasmid, and now I want to clone the spliced variants into plasmids, so I can express them individually in different lines.
How can I get rid of the part that is spliced, and re-connect the two desired fragments, accurately and without adding anything into the insert?
This is what did not work, so far:
1. I can't use restriction sites because they are not accurate. I need to take out a very specific fragment, not a general area.
2. When doing PCR to the whole gene, it only works when the template is the plasmid, and than I don't get the splv's. If the template is a cell line or a tissue sample, I dont get bands when using primers to the start and end points of the gene.
3. I generated the two desired fragments, using specific primers, where one of the inside primers has a flanking region that is complimentary to the other inside one. I received the two fragments, but the completion reaction, when they should connect, doesn't work.
4. I dont think it will be right to insert a restriction site into the middle of the gene, which is what will happen if I use one to re-connect the fragments.

What else can I do? How do I get a specific area out and re-connect the two sides???

Thank you so much for your advice,

Shulamit.

I think you can tried to optimize the PCR condition or switch to another Taq and amplify the splice variants from cDNA directly.