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 don’t 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 don’t 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,

Shu.

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 don’t 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 don’t 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,

Shu.

Hi.

2. You said that simple PCR on the sample gene (RNA presumably?) doesn't work, so how did you get the full length clone? If you obtained it elsewhere your problem may be just too long PCR product or low abundancy. Try some kits for long-PCR if you can, but in mRNA it all depends on the abundancy of the transcript and the quality of RNA, getting longer products is sometimes very tricky. You have whole RNA or just mRNA? mRNA fraction would have higher percentage of your transcripts, maybe even worth to try experimentally increase the transcript of interest before isolating. (this only applies if you can get a fresh RNA, from the cell-line or so)
3. The the completion reaction, how did you do it exactly? (I would imagine a one step denaturation, and elongation step with a forward primer to fill in the missing 5' end, then separation on gel). Or wouldn't be possible to have two perfect "halves" and then blunt ligate them and separate on the gel?
4. There are transcripts bearing a artificial restriction site in the middle (of course not shifting the reading frame) and still working, but you'd have to prove experimentally, that the protein is the same. I recomend rather trying something above.