I am trying to clone 2461 bp fragment (restriction enzyme sites are included) into pET28a+ plasmid (which has his-tag that i will be using for purification of protein of interest later). So far, amplification of gene and digestion were completed. Then for ligation step, different V:I ratios were tried and these are 1:1 1:2 1:3 1:5 1:7. Not a single colony was obtained after transformation of ligated product into competent E.coli DH5a. This cycle was repeated multiple times.
What should i do after this point? Is the fragment too large for this vector or can i try a different V:I ratio again or should i use a commercial cloning kit? If so, which kits can be used in this case?
P.S: I have recently used these restriction enzymes and competent bacteria for another cloning experiment and there is no sign of contamination or faulty.
Hi Eka,
2500 bp should not to be too large.
I guess you have checked the quality of your DNAs by gel electrophoresis, that your restriction sites are unique and your primer have additional 5'-nucleotides preceeding the restriction sites?
Alternatively, you may switch to recombineering, e.g. Gibson Assembly, or clone your PCR fragment buntend into a cloning vector, followed by sequence verification and transfer to a pET-vector.
Best,
Uwe
I would also suggest sub-cloning your fragment first. The pTOPO vector system can capture either a blunt or A-overhang fragment (TOPO blunt or TOPO TA vector). That way you can check each of your restriction enzymes (ensure they are cutting your fragment ends) and keep a nice available stock of your fragment so you don't have to resort to PCR amplification every time you need more. You can sequence the fragment in pTOPO as well to make sure you are moving forward with an accurate copy to the final vector.
Hi Ece,
The cloning procedure you described should be fine, especially if you validated your reagents with another cloning experiment.
I would suggest to try homologous recombination-mediated cloning. It is very easy and cheap (basically you just need to amplify by PCR both the insert and the plasmid with primers that introduce homologous sequences, then mix the two amplicons and transform competent cells).
You can find detailed information on how to optimize the procedure in this interesting paper: Article Optimal Cloning of PCR Fragments by Homologous Recombination...
This helped me several times when traditional cloning by restriction digestion and ligation failed.
If you have repeatedly attempted conventional cloning with different vector-to-insert ratios without obtaining any colonies, there are several alternative methods you can consider for cloning your 2461 bp fragment into the pET28a+ plasmid. Here are a few options:
Before switching to an alternative method, it's a good idea to verify the integrity of your insert and vector DNA by running them on an agarose gel. Additionally, you may consider performing a negative control ligation reaction without the insert to rule out any potential issues with the vector or ligation reaction.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
If you are digesting the PCR amplicons directly, do ensure that you leave 3-5bp overhangs after the restriction site. Most restriction enzymes require 2-5bp away from the restriction site to sit on and cleave the DNA. This is why first cloning into a cloning vector like pCR-TOPO would be a good idea.
you get il all with Nikolay Klyashtornyy and Ananth Krishna Narayanan answers
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