I'm trying to clone a ~900 bp gene which has a high GC ratio (about 75%) in E. coli. (Top10). The thing is, I'm not sure if the ratio of GC is a criteria in choosing the vector or rather, if there are specific plasmids that are more suited to clone of these sort of genes. Any suggestions?
Thanks.
- from which organism does your gene come from?
- what do you plan on doing? overexpress in bacteria? protein expression?
Dear, it is from human genome, I,m plan to sequence this fragment at first and to do protein expression later.
For sequecing you can use a vector for PCR cloning like pGEM, pJET or Topo. But for protein expression you may encounter some difficulties since is a human gene expressed in E. coli. You will have to check for glycosilation sites, dissulfide bond formation, solubility, etc.
Dear,
I tried for sequecing a vector for PCR cloning pGEM, pJET, I hadn't any result. do you think I must use a special ligases enzymes for ligation?
Did you confirm your cloning by restriction enzymes?
I believe the problem was the method you used for sequencing. Did you use Sanger? There are enhancer solutions that are suitable for rich GC inserts. Thermo Fisher sells SequenceRx Enhancer Solution A that helps with inserts like yours.
The GC content will not affect cloning. Are you using Taq or Pfu?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning