I am trying to clone a gene of 2kb size from one plasmid to another. I first did a pcr for my insert gene from plasmid 1 and then gel elution of amplified product. I then did a single digestion in plasmid 2 and insert and then ligation and transformation in DH5alpha E.coli. I got the colonies. But I had almost equal number of colonies in my -ve control as well (religated plasmid without insert). I then randomly picked colonies and did a colony PCR with primers between vector backbone and insert, for which I got positive result (200bp). I then isolated plasmid using qiagen miniprep kit and then did a pcr again with primers for my insert gene and now I am getting a product of size 800bp instead of 2kb. Then I did a test digestion of the isolated plasmid and, no insert. There was no contamination in PCR reagents. The primers don't have any other match in the plasmid. The annealing temperature was 67 (Tm of the primers is 74). The restriction endonuclease has high fidelity. Can you please explain what went wrong and should I continue once again with a dephosphorylation of vector before ligation?
First, even though you gel elute the PCR product, that mixture will still contain some plasmid DNA from plasmid 1. If there is sufficient plasmid 1 around, it will show up as colonies when you do your transformation (assuming it has the appropriate selectable marker). Generally, if I do not see a significant increase in colonies over the negative control, then I do not bother to screen colonies. This is because I always get screwy results and I end up wasting time. Try to optimize your ligation to give the expected increase in colony number after ligation with your insert.
Do not treat your cut vector with phosphatase. It will remove the phosphates from the vector. If your primers used for PCR of your insert do not have 5' phosphates (they normally do not unless you request it specifically when they are ordered), then your ligation will not work.
If primers used are normal, then the PCR products would have 5` phosphates. So, using a phosphatase would definitely help. Otherwise, there is also high probability of your vector re-ligating.
Did you do a colony PCR from the negative plate as well? I think it would have also given a band as your positive plate.
Challa, It is a cohesive end ligation done in the same way mentioned by you. David, since I am also going to restriction digest the ends of my insert, I will have 5' phosphate in my insert. So, I can do phosphatase treatment to my vector and ligate. Sneha, thats a good point, I will do colony PCR for my negative plate.
i think your gene is not inserted into the plasmid. I mostly used CIAP (phosphatase) treatment for single digestion-ligation to avoid self-ligation.
It is obvious that you don't have insert in your plasmid. I would suggest you to optimize ligation by trying different plasmid: vector ratio and ligation time. It is most probably that you had re-ligation so if you can use white blue colony selection that would be very helpful to know which colonies to check... Anyway you can also simply check isolated plasmids on agarose gel without digestion and if you have your insert inside you will notice shift on gel, then you confirm that by digestion and sequencing.
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