I am using two enzymes BamH1 and Not1 to cut my insert and vector both.Aftr transformation,I am not getting any getting?
Do Bamh1 not1 are not compatible ?
Hi
Unsucessful transformation can be due to alot of reasons: Below are a few of them
1. Digestion inefficiency: If the REs you use are not of the high fidelity class, then you may have to use a buffer that is compatible to the two enzymes. If you are using thermoscientific REs, Bam HI buffer can be used but with two fold of the notI enzyme. Alternatively, you can use buffer O but with 4 fold excess of BamHI enzyme. Please use the link below for your guide on double digestion
https://www.thermofisher.com/my/en/home/brands/thermo-scientific/molecular-biology/thermo-scientific-restriction-modifying-enzymes/restriction-enzymes-thermo-scientific/double-digest-calculator-thermo-scientific.html
2. Ligation failure: If your ligation reaction is inefficient, even with good digestion, you might get nothing after transformation. To rule out this, you can do a control ligation reaction.
3. Loss of competence of cells: are your competent cells commercially obtained or you prepared them in the lab? have you tested them before? How old are they in your possession? You might wish to do a control transformation using a known plasmid to make sure your cells are fine.
It will however be more helpful if we know the specific vector you are using and whether your insert is a cloned gene or a PCR product.
Goodluck
Bashir
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