I did cloning. it was like a traditional approach. Firstly, Restriction digestion was performed for both vector (8kb) and target gen (700bp), and also the vectors and target gene were run on the gel, cut and purified. Then, the gen of interest was ligated with the ratio of 1:3 into the vector. After the transformation, bacteria were cultured on a LB agar plate overnight, and I saw about ten colonies on the plate. Then, three single clones were picked up and grew in the LB+ antibiotic broth for 18h. Miniprep was performed and ran on the gel (1%), but I didn't see any band for the target gen. There was only one band for the vector. I can't understand why the bacteria could grow on the agar plate while cloning never happened. Also, two sticky ends of vectors made by two restriction enzymes are not compatible. The question is when you have clones on the agar plate, It doesn't mean that the ligation is successful? please let me know how i can optimise this?
I have also attached the image of LB agar plate after cloning
Neda Mohammadi I suppose you used LB+Antibiotic agar plate to select cells containing the vector+insert of your interest. Can you clarify if you have done a restriction digestion after miniprep? If not, you are going to see only a single band corresponding to the vector+insert.
Yes, I used LB+Antibiotic agar plate to select transformed bacteria. I also did restriction digestion after miniprep and then used agarose gel to check cloning.
Neda Mohammadi May be you did not digest for long enough? Or you digested a lower concentration of the vector+insert. Your insert is still in there, you can not see the lower band due to lower concentration. Compared with your insert, your vector is at least 10 times bigger and you would expect a super bright band compared to your insert on a gel
You can simply do a PCR with the miniprep to confirm the presence of insert.
I am quite sure that the digestion was pretty enough because this vector had another insert which was digested with the same enzyme and. Then cut vector was run on the gel, and I could see two bands (one for the vector and another for the gene of interest).
In case of low concentration of the vector+insert, you mean the vector might be from the low copy number class? Because when you increase the number of bacteria by culturing in broth, It means that you also boost the number of vector+insert.
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