I tried to ligate my insert (520bp) into TOPO vector (TA cloning).
My PCR product has concentration of 30~40 ng/mcl
I used the mixture as below:
PCR product 3 mcl
TOPO vector 0.3 mcl
Salt 1 mcl
D.W 5.7 mcl
Incubate at RT for 30 min
Than transfer the mixture into Competent cell.
However, I just got the blue colonies, without any white colony. I did try 3 times with the same results.
Previously, I did the same protocol for another construct (625bp), The results were good.
I don't know what could cause the problem for this new construct...
Hi Andreas,
In the protocol for TA cloning using TOPO vector, they do not suggest to use any ligase ...
Possible reasons
1. There is no TA overhang either because you used proof-reading enzyme for PCR or you waited too long after PCR to do the ligation. The fragments tend to lose the overhang with long term storage.
2. Some DNA fragments are hard to clone; either they are unstable or are toxic to bacterial cells.
Doesn't the TOPO cloning protocol ask for 1 ul of the vector and 1 ul of salt solution and a total reaction volume of 6 ul (unless the protocol has changed recently)? Your total volume is 10 ul and therefore the final salt concentration is not optimal. Also, 0.3ul of vector may not be sufficient enough. You see, in your reactions, all the concentrations are way off.
Thank you, Ambady.
You are right about the protocol of TOPO cloning kit. I just followed the modified protocol of my senior. I am going to try and follow the manual of the kit...
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