I am doing gene cloning for the last two weeks. I have doubts that my ligation reaction is not working properly. Kindly suggest how to validate it.
Hi Praveen,
Sry to hear that! How do you do the ligation?
I usually do it overnight at 4°C and in difficult cases I add some PEG (15% PEG (MW 6000)) to improve ligation efficiency.
Good luck ;)
Hi Praveen,
I tend to just leave the reaction on the bench (usually around 20 C) overnight. They do recommend 16C or 4C to make the fragments to be ligated less mobile and thus facilitate the chamical reaction of the ligation. I have tried both way and have not found much difference, but that's just my experience. Maybe you could do both in parallel and see whether that does it.
However, also make sure your restriction enzymes are inactivated and/or your insert and vector are clean and pure.
For ligations the relative amounts of vector and insert are crucial for it to work. Make sure you run your cleaned pieces on a gel and looking at the bands you decide how much of each you put into the reaction. Usually 3:1 insert to vector does it, but you can try different ratios because these will depend on the size of the insert, the bigger, the more difficult.
I hope that helps
Hi Praveen,
I suggest you check the reliability of your Ligation buffer. Multiple freezing and Thawing results in degradation of ATP. Try borrowing the reagents from the lab next door or you can even add ATP (1 mM) to the ligation mixture. 4 C overnight is usually followed for convenience but 10 mins at room temperature works very well for me (i use T4 DNA ligase from Thermo).
all the best.
kind regards,
Rabbind.
- Badges
- Science topic