Hi Everyone. I am new to molecular biology. I am currently doing ligation and transformation. What I did was I did a blunt end ligation of my inserts which is 1257 base pairs to my vector which is 3300 base pairs. I had previously digested. My vector (pComp3xss) with sfiI and that I used the 3300 basepairs fragment (vector) to ligate my insert. I did a 1:3 vector to insert ratio. However when transforming, I had no colonies. But for my positive control, the uncut vector, there was growth. I used a ligation protocol from thermofisher however I did not use PEG as states in the reaction protocol provided. I only used T4 DNA ligase, T4 DNA ligase buffer (10x) and water. What could be the problem? Thank you.
If your insert carried blunt ends and your vector was digested with SfiI then the ends are not compatible and won't ligate.
When you said you had colonies with the positive control (uncut plasmid), how many colonies did you get? Your plate should be nearly confluent with many thousands of colonies, is that what you got?
Hi Michael. Thank you so much for replying. I did treat my vector that was digested with sfiI using an End-repair mix. And than ligated both the insert and vector using T4 DNA ligase. For my uncut plasmid which is a positive control, I got a bacterial lawn rather than colonies.
It sounds like your transformation was good at least. So it comes down to either your End repair did not work all that well or your ligation conditions are not optimal. Note that blunt end ligations are much less efficient than sticky ligations.
Before getting too worried about it, I would just repeat the experiment, be sure you have plenty of both vector and insert DNA going into the ligation.
Assuming you did not dephosphorylate your vector, there should be significant background of the vector recircularizing.
At what temperature are you putting up your reaction and for how many hours?
when i put up a reaction with t4 , i usually keep it at room temperature for 3-4 hours and then overnight at 4 degree.
next day i transform the ligation mix
Thank you so much. I have read about blunt ends ligation. That it is less efficient. I will definitely try ligation overnight today and do transformation tomorrow.
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