Hello all,
I cut my 13.3 kb plasmid with EcoRI and XhoI. I didn't perform phosphatase treatment and continued directly with gel purification step of the plasmid. I tried to ligate a 114 bp insert to my vector plasmid (13.3 kb) with 1:1, 1:3 and 1:10 ratios. I used 100 ng of vector plasmid in each 20 ul reaction. I performed o/n ligation at 16 C and transformed the whole ligate (all 20 ul). After that, I didn't see any colony formation on my agar plates. What could be the possible reasons?
One thing what you can do would be to check the activity of your ligase. Just use a standard plasmid, cut and religate it, you will see if your ligase works. If not, sometimes increasing the temperature up to 22°C may also help.
I hope this will help you to solve your problems.
Thank you very much for your answer Justin. I already repeated the reaction again and transformed 5 ul of 20 ul reaction. I hope that it works. And I will keep your suggestions in my mind.
Thank you very much for the suggestion Can. As I said, I already tried different transformation. Let's see. If this doesn't work, I will definitely try your suggestion.
Hello Aysegul,
In my opinion, the digestion didnt work at all. Which buffer did u use to do the digestion? From New England Biolabs, it is recommend NEB3.1 buffer. You might incubate for along time (~12-16hr). Or sequential enzyme digestion.
114 bp is small, you should make sure after PCR purification you have high concentration of your insertion.
With ratio 1:3 - I think you can have successful ligation in case the plasmid is 90-100% cut.
Hi Trang,
Thank you very much for your answer. I usually incubate the sample with REs either for 4 hours at 37C or O/N. I used cut smart buffer actually. Because one of them is high fidelity enzyme. Let's see. I am trying the digestion one more time. Transformation didn't work again.
Using Smart buffer is said it is perfect for all of digestion. However, in some cases using smart buffer will affect the digestion reaction.
Could you reduce the amount of plasmid you are using. From the gel, it seems the plasmid is too much.
Are you sure the competent cells work well?
Another suggestion, why dont u try another set of enzyme digestion? When I use EcoRI and XhoI in the lab, my cloniing also didnt work, then I try another set of double enzyme digestion.
Dear Trang,
Thanks again for your feedback. I didn't know most of them.
Yes I am sure that competent cells are working fine. Because all my colleagues and I are working very well. Unfortunately, I can't use another REs. Because this is the only place I could clone into my vector.
I understand the situation. Could you try the following changes? I hope it will work for you.
- Let reduce the amount of plasmid around 5 times (based on the gel photo)
-Increase the amount of insert.
-Try NEB3.1 buffer instead of Cutsmart buffer. Cutsmart buffer causes star activity for EcoRI.
The below is the suggestion from the NEB company.
https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder?enzyme1={2F6573A8-32BE-44A3-A0B6-C2089082ED9B}&enzyme2={69E7E307-0F85-40E0-85D1-1114E13B0B18}
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