I want to insert my recombinant DNA of about 1650bp into pET22b which is about 5500bp. The resultant product from the ligation will be used for E. coli transformation. I seem not to get a positive result with the protocol I am using. I would be grateful if anybody with experience this area could help me out.
what is the problem : No colony at all or lots of clones with no insert ?
You should trouble shoot each step of your procedure to see where it is failing. For example, be sure you have sufficient good quality DNA of the insert and the vector. Next be sure each restriction enzyme is actually working well. Then confirm that your ligase and ligase buffer are still working well. Lastly make sure your competent cells are good and you get high efficiency transformation with an uncut control plasmid.
I agree with Michael. Since no colony on negative control , more probably ligation might be the problem. Use new ligase buffer, keep it cold and vortex it well before use . To check ligation, I would have single digested plasmid as positive control
How much DNA do you have to work with and what are your downstream applications. Do you want a double digest or two individual digests. There are a few other methods that you could also use.
Thank you all very much for your contribution. @Michael, i will get back to you with the feedback.
For any double digestion, first of all you have to select a suitable buffer and temperature for restriction digestion that is optimum for both the enzymes. If not, you may need to perform the digestions at two steps depending on different buffer and temperature requirement. If you are using, NEB enzymes, nowadays they have online tool to identify the buffer and digestion condition. For the present situation for BamH1 and Not1, you may use NEBuffer 3.1 at 37°C. I got the same from below link:
https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder?enzyme1={4EC4ABBB-EF3C-4582-ABD3-367A0DA68748}&enzyme2={6D1004FC-499F-4BDE-9227-4FA21E0D2179}
If it is step-wise digestion, then one should be careful of completely removing residual chemicals before second digestion. Also, this can be done first with low salt containing buffer and then only high salt containing buffer at specific temperature. Another important point to be taken care is the ligation conditions (timing and temperature) including the fresh ligation buffer and the DNA ligase enzyme should be active. Good quality purified DNA of 1 : 3 ratio (50 ng of vector : 150 ng of insert) should be used.
And make sure that Noti and BamHI do work in single enzyme digestion reaction.
Based on my experience i can say that NotI cloning is a bit more tricky.
HI Desmond,
Please be aware that If you use 50 ng of the vector and 150 of insert while the ratio in weight is 1:3 the molar ratio will be about 1:10, which COULD promote multiple insertions.
https://www.neb.com/tools-and-resources/usage-guidelines/tips-for-maximizing-ligation-efficiencies (look up the DNA section for molar ratios)
Since you do not know exactly what your problem is I would follow those guidelines strictly until you figure it out.
Cut both vector and insert with BamHI and NotI in NEB 3. Use 1:5 vector/insert ratio. This should work. If you wish, you can sequentially digest the vector and insert before ligation. Good luck
@samir..u r saying that not1 cloning is tricky.
What is the reason behind this?
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