I am currently constructing plasmids for protein expression. I would like to seek for your suggestions on how to troubleshoot my failure on constructing one of my plasmids. The trials that I have made will be elaborated below for better understanding.
I would like to clone my gene interest, which is 2.5kb long into a vector, which will be 5kb long after digestion. Firstly, both vector and DNA inserts are incubated with two restriction enzymes, Ecor1 and Xho1, at 37˚C for 2 hours. After digestion, the digested genes are cleaned up whereas the digested vectors are ran on a gel for purification as well. Then, purified genes and vectors are ligated at 1:2 ratio of vector:inserts using LigaFast T4 kit. According to the manufacturer’s protocol, reaction mix is incubated at room temperature for 5 minutes. Transformation is then carried out by adding the 20µl of ligation mix into 25µl of TOP10 competent cells. Above is my very first attempt.
After the first trial, further attempts were made by changing conditions at different steps and remaining steps remain the same.
Attempt 2: 3 hours restriction digestion of vector and DNA inserts.
Attempt 3: Incubate ligation mix for 10 minutes, 4 hours and 16 hours at room temperature.
Attempt 4: Incubate ligation mix at 4˚C, 16˚C and 37˚C for 16 hours.
Attempt 5: Prepare ligation mix at 1:3 ratio of vector:inserts.
Please share your opinions or your experience with me. Your reply is much appreciated. Trying so hard to get my final construct ready.
Are you getting any colonies at all? First thing I noticed is that you are adding way too much ligation mix to your cells. Reduce the amount of ligated DNA mixture to 1-2 ul. You can run 10 ul of the ligation on a mini-gel and check if the DNA actually ligated and changed conformation. Second, if you are getting colonies but the plasmids have no inserts, then you are getting vector self-ligation, which can be avodied by treating the vector only with calf intestinal phophatase which removes 5'-P so the vector cannot self ligate. Third, do not spend a lot of time trying to change ligation conditions, these are pretty standard. Room temp overnight should be plenty of time, you cannot over-ligate. Check all your DNAs on a mini gel before and after ligation and you should see a difference.
Hi, Mr. Gregory Dressler. First of all, thank you for your answer.
Some attempts that I have mentioned did give me few colonies but my inserts are not detected. When you mention check DNA before ligation, do you mean to load the ligation mix or just the digested vector alone?
Alright, I will load lower volume of ligation mix for my next attempt.
You can check the ligation mix before and after ligation to see if there is any differences, as any ligated products will run differently on an agarose gel. Of course you need to ligate enough to see on a gel. If you use 200 ng of vector and 400 of insert, you should easily see this even if you just run 1/4 of the mix before and after, saving the rest (1/2) for transformation.
- Badges
- Science topic
- Similar topics
- Comment