Hi all,
I anticipate that I am completely new and for the first time I have to do cloning of genes whose sequence is unknown (The final objective is indeed to sequence the gene). Since I am not supervised at moment I am quite concerned about it . I read up about cloning but some aspects of what I am supposed to do are not clear. For example, based on what do I choose a restriction enzyme suitable for my gene if I don't know the entire sequence of my gene? (so far I just sequenced a fragment amplified by PCR to be sure that my bacterium had the gene I was looking for, but I need to sequence all the gene in order to see also the genetic environment of it ). I don't want to risk that the enzyme cuts inside my insert.I thought to take the sequence of a known gene that is homolog to mine and check which enzymes cut into it so as to exclude them. But since my gene is probably inserted into an integron whose sequence is again unknown how can I be sure that the enzyme does not cut into it?
I hope to have been clear.
thank you in advance
If you really wish to generate a genomic library to pull out your gene of interest, then traditionally this would be done by using an enzyme that cuts frequently and doing a partial digest so that the genome is fragmented somewhat randomly. You can review any "Molecular biology" lab manual for how to construct a genomic library and adapt for your situation.
On the other hand, if you have partial sequence then you could try just doing inverse PCR and sequence the products to generate the sequence upstream and downstream of your ORF and then create primers for amplification.
Lastly, given the low price of sequencing bacterial genomes, you might consider just sequencing your entire genome and save yourself weeks or months of work.
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