Hi, I am trying a cloning over a long time. My vector is of about 4.7 kb and insert 1.4 kb. I am using Xho1 and Kpn1 as restrictions. I am attaching some files for details. In the first image I have digested individually the vector with two restriction enzymes, where Xho1 shows multiple bands. In the second image I have ligated and un-ligated products in the first and second lane. In the third image I have the ligation mixture transformed, the problem is I am not getting plasmid from the colonies. Are this colonies fine or they are non specific ?
the colonies on those plates look fine. If they are growing well then you may have an issue with your minpreps
The ligation procedure seems fine, however, I disagree about the colonies looking normal. There is a large variation in your colonies size (this is an indicator of toxicity).
the variation in size could be for two reasons:
1-the plasmid is toxic or unstable (in this case either use stbl3 cells or nebstable and use 30C for culturing procedures)
2-You are using home-made competent cells and taking them at a point where not all the cells are in the same phase (in this case the variation in size is fine).
I would suggest you use the competent cells mentioned above, I prefer stbl3 out of the two, and repeat the transformation but recover and plate at 30C then selected the smaller colonies on the next day.
Looks like a straightforward cloning. However, the pictures need markers, so I can judge the size of the fragments on the gel. Also, indicate what is in each lane. Check also whether both enzymes are cutting.
As for the colonies, there is always a small number of colonies without the desired plasmid. In crease of the concentration of antibiotics will make the a-specific colonies disappear.
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