Particularly, I have a 14kb mature transcript that could make for some exciting biology experiments. The current plan is to create a cDNA library and amplify ~4kb parts of the GOI and use endogenous restriction sites to ligate them together into my transformation vector. Does anyone have experience in cloning large genes and would like to share some helpful insight on this process? Is there a better way? I feel as though the RT-PCR process might be the most troublesome with this approach, so any suggestions about that would also be greatly appreciated.
We have experience up to 10KB genome of an RNA virus. Initially we have sequenced entire genome by overlapping PCR and designed primers to amplify around 3 kb fragments and ligated them one after the other. It takes long time. After sequencing the full genome clone we found some mutations and we are trying to get synthetic genes. I think going with synthetic genes may be less time consuming and cost effective. Here you can mutate the gene to create new restriction sites.
with increased size of the gene to be cloned availability of restriction sites will become less. The other alternative is using ligation independent cloning (LIC) methods. Kits are available for LIC.
Best
Hi Laurence-- This is all really good information, thank you for taking the time to write it all!
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