What is the best temperature and incubation time for successful ligation of blunt ends?

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There is no colonies after Ligation reaction and transformation ?

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Robert Adolf Brinzer

Have you tried adding 2% PEG? What concentration of vector are you using? Did you only dephosphorylate one of the two fragments?

Alexandra Johnson

What results are you expecting vs. what are you getting?

Amy Klocko

The degrees and times you are using are reasonable. I agree with the Alexandra's suggestion above to share a bit more of your results. Are you getting any colonies at all? What are your controls?

A few things to check are your buffer/enzyme compatibility (just make sure the buffer is the one meant for that particular ligase and is used at the proper concentration) as well as the expiration date of the buffer (they do expire, and the ATP will degrade over time and with freeze/thaw cycles).

Mohd Akram Ansari

I have done it at 16 degre centigrade for overnight. I would suggest please go through manufacturers instructions for ligation procedure.