Hello, I am currently making a luc gene.
The primer was ordered and the band amplified with pcr was gel extracted.
Restriction enzyme was treated to make the insert sticky with the vector... Hindlll was cut into 2 cuts in the insert, and it was cut in the middle.
I want to cut only the end, is there any way?
For example, the restriction enzyme treatment time, or control the amount?
Sorry for poor English. Any help would be appreciated.
Hi there,
I think the best is to consider using restriction enzyme which is actually not cutting into your DNA of interest.
Dominique is right. You might manage to clone using partial digesting but at the end you are probably going to cut your insert back from the vector and that will be difficult.Just redesign the primers for a new enzyme site and re amplify some of your already amplified pcr product.Primers are cheap and it will save a lot of time
Hi,
If you have 2 different restriction enzyme sites in your Fragment you may can digest your fragment with one enzyme and make one site end sticky with vector and other blunt end (instead HindIII). Thus, you can clone into your target vector with one end site EcoRV that is blunt end as well. On the other hand, you can clone your fragment in a TOPO or pGEM vector and make the digest with a alternative enzyme present only in the vector and transfer your fragment to your target vector. If these two options will not work for you then you should design new oligos with different enzyme sites that do not cut your cDNA. Best wishes.
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