Hi Fellow Scientists!
I am trying to clone my 300 bp insert into a 6 kb vector. I run a few different ligation reactions using (1:3 vector: insert molar ratios) 10, 20,30, and 40 ng/ul vector amount in a total 20 uL reaction volume. I am using 50 ul of DH5 alfa competent cells (Thermo). I'll do the transformation, but I am not sure how much DNA should I use for the transformation? Should I use 2 ul from the ligation mix as recommended in the manual or should I use more? How much DNA can these cells handle and what is the required amount for optimal transformation?
Any help much appreciated!
Thanks in advance!
Hello, better to use 2 ul from the ligation mix as recommended in the manual but normally the form 1 to 5 is suitable.
Hello, first did you get any colony using your conditions?
if not you can try 1:5 (vector: insert) molar ratio using 200 to 500ng total amount of backbone. Then you can transform up to 5uL in your 50uL aliquot (should not exceed 10% of volume of cells).
Hope this helps.
Try following the protocol that came with the ligation kit, then run out some of your ligation reaction on an agarose gel compared to the vector. The protocol will also say the concentration of vector to use and the minimal and maximum amounts. You don't need to set up every possible ligation reaction, just pick one that is within the standards.
Use the 2 microliters of ligation mix that is recommended in the manual. The amount of DNA is not the rate limiting step in transformation.
Remember, your goal is to successfully transform your cells with your plasmid so you can continue with your actual project. You are not trying to optimize a ligation & transformation protocol. Write down what you did, how well it worked, and keep going with your research.
Thank you so much for all the useful answers! I tried 5 ul as suggested by most of my friends and colleagues and no colonies at all.
Karen A. Darbinyan
I tried before 1ul, 2 ul, and 1:10 dilution of ligation mix for transformation, but could not get any colonies.
Martin Pelosse
I also tried 1:5, 1:10 and, 1:15 (vector: insert) molar ratio using around 100ng total amount of backbone. However, I used the amount that I mentioned above for transformation. Since this time I used a lesser amount of total DNA in the ligation I used more (5 uL of ligation mix) for transformation.
Katie A Burnette
I can't see anything on the gel when I load the ligation mix. This is raising the question that whether my ligation works or not. I could be able to see it on the gel, even it's a ligation reaction, right? I set up different reactions by using different concentration of vector that falls between the min-max amounts just to get a colony. None of the reactions did work. At this point, I really don't know what should try more or what I am doing wrong.
As a side note: I know my transformation works, cause I did transformation using puc19 control and got colonies. Also, I can get colonies if I use uncut plasmid (10 ng) for transformation.
Any further help is much appreciated!
OK, sounds like the ligation isn't working. You should be able to see your vector and your insert on a gel (and the ligated vector + insert product).
Use a ratio of 100ng(Vector):200 ng (Insert)... Adjust the ligation buffer and Ligase enzyme according to total volume. Give an ovenight incubation at 14-16C before transformation (You can use all ligation mix for transformation). But remeber it may happen because of improper restrictions too. Sometimes either or both of your enzyme is not working properly. So try restriction with different set of enzymes if your ligation still fails.
- Badges
- Science topic
- Similar topics
- Vectorization