Hello everyone, I hope you are very well.
I am performing site-directed mutations with the QuikChange II kit. My first ones have a length of 50bp and a Tm of between 75 and 81 approximately. I have followed the kit protocol, both for the mutants and for the control plasmid that comes with the kit.
The strange thing is that after the transformation I do not obtain mutant colonies or colonies with the control plasmid.
Before digestion, I get bands on the PCR.
At first, I thought it was a problem that the plasmid to generate the mutants would have been grown in other bacteria, so I took the same ones from the kit and obtained several colonies, purified the plasmid, and started all over again. I did not get colonies.
My concern is that I am not getting colonies from the control plasmid, I don't know if I am doing something wrong or what is happening.
We have even used other dNTPs and still have no results.
Is there someone who can guide me?
Dear FRancisca
if you are not see colonies also for your control plasmid it seems that you have some issues in the transformation step.
Is your kit and your competent cells old?
However for the future i would uoi like to suggest you to use the PIPE cloning approach instead the Statagene KIT.
THe principle it is the same but you do not need yo use any specific kit, but just a high fidelity polimerase to amplify the vector and the mach1 competent cells for the trasformation.
Attached you can find some article about it.
Yuo can find more information also in my blog: ProteoCool https://proteocool.blogspot.com/ at PAGE1 (cloning)
ciao
Manuele
I had the same problem using this kit, although I got colonies from the control plasmid.
1. I increased ul of Dpn1-treated DNA, so I transformed 4 and 6 ul of the mutated plasmid, instead of 2 ul reported on the protocol.
2.Then, I plated 300 ul rather than 250 ul.
In this way, I obtained around 10 colonies from the plate with the mutated plasmid ( better than nothing) that I further amplified by maxi preps.
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