Hello everyone,
I have been trying to clone some genes into vector plasmids i have created. using a single restriction enzyme to digest my circular plasmid.
I am following a path like " restrict - gel purify - dephosphorilate the vector only - heat inactivation of phosphatase - ligation - transformation"
I am having enough colonies but somehow no insertion and I am observing multiple bands after colony PCR. some colonies still has the right size dense band but sequencing says that its empty...
I have tried to play around with the vector:insert ratio, but, it did not help.
Would anyone have some suggestions on what to improve? I would also appreciate improvement on how to dephosphorilate my vector.
Thank you,
Metin
regarding the amplification of insert but there is no insert in the vector I think that you may be using primers for the colony pcr that are both inside the insert.this means that even when the cloning has failes the wetting effect of reactuin mix on the agar plate allows pcr amplification. The safe way to test for insert is to have one primer in the insert and the other prier in the vector then a positive pcr must be insert inside the vector
@Paul
Thank you for your answer. For the colony PCR, I am using one vector and one insert primer.
If you have some colonies that by PCR or plasmid mini prep analysis seem like they are correct, but not by sequencing, it may be that you have a mixed plasmid prep. You could try and retransform with some of that mini prep at very low DNA concentration and then analyze some of those colonies to see if you can find any that are correct.
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