Hello everyone, I am currently working on a clonning experiment where I inserted my gene into a special vector and transformed it into DH5alpha competent cells. The problem is after translation when I checked the petri dishes I saw that the amount of colonies on both experimental (the ones that I estimate that have the insert in the vector) and negative (blank vector) dishes were about the same. I think the ratio should be at least 1/5, so I immediately thought that something went wrong at the ligation phase. I know that my vector is working well because other lab partners also use it, so I believe my ligase was problematic(somehow denaturated or something) or the buffer I used was out of date (How can it be possible??)
Those are the first problems that came to my mind. Addition to that, what do you think might have happened? What are the other things that I need to check to detect the problem correctly?
Kindly thank you for your answers.
Hi there,
What is your control? Plasmid alone or plasmid with ligase (actually you should run both of them). If the former, your plasmid is clearly not properly digested. If the latter the plasmid is not digested and/or selfligates. Which means the event you are looking for is rare compared to the selfligation event. Only the comparison of these both controls tells you about the frequency of non digested vector and of selfligation.
A simple explanation for failed ligation: the insert has no compatible ends with the vector.
What is the size of your vector and your insert?
In the mean time try ligation PCR with either vector specific primers or one vector and one insert specific primer. Ligation PCR requires as little as 1ul of ligation mix. This will ensure that ligation has worked.
Also, make sure you're using fresh LA plates with appropriate antibiotic. Try plating only comp cells to check if the antibody is working or not.
1- depending on the size of V and I u choose the ratio and 1:3 is the best
2- incomplete digestion vector increase background
I am not sure what is main issue u r facing but I can tell multiple problems maybe and if you clarify them more it will be easy to resolve.
It also seems that the vector is religating as you are getting similar colony number in your test and control plate. SAP treatment of your vector after digestion should help as it will prevent religation of vector.
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