General setup
- 250-500 ng vector
- 5-20x (in molar ratios) of insert--higher insert:vector ratio for shorter inserts
- 2 ul T4 DNA ligase buffer
- 1 ul DNA ligase
- I have good luck using less ligase (say, 0.25uL per reaction). -Josh
- Water to 20 ul total volume
- For routine cloning, I also run ligations smaller than 20uL. I've never understood the reason to run a 20uL ligation, transform 1uL, and throw away the rest. 10uL is fine, and you use half as much ligase. You can go to 5uL if you want to push it. -Josh
Reaction time
- According to NEB
- At room temperature, use 1 ul of ligase in 20 ul rxn; 10 min for sticky end and 2 hrs for blunt end
- At 16C, 1 U of enzyme can ligate 50% of HindIII fragments (at 300 ng/ul) in a 20 ul rxn in 30 min
- Room temp, >10 min. I'm not aware of any reason that running longer is bad. I tend to run my ligations as long as is easily practical. -Josh
- Room temp, 3 hrs (YC). I have tried transformation after a 20-min ligation and know that it yields fewer colonies than a 3-hr ligation. However, if you generally get more than enough colonies to screen, then the shorter ligation time may be sufficient.
- 16C, overnight (YC). I tried this as the last resort after multiple failures in cloning 3 constructs. 1 of the 3 constructs worked (and I'm not sure it's entirely due to the different ligation protocol), while the other two constructs showed no significant improvement (one went from no colony to 2 colonies; the other from 2 to 3 colonies).
- I think overnight ligations are useful when you're doing tricky cloning. Blunt end, for example, or when you're cloning a library (and need lots of colonies). -Josh
Concentration and purification after ligation
- In general, I (YC) do not purify or concentration my ligation products. In the one instance where I did, I simply got 40 instead of 3 negative colonies, suggesting that I was concentrating a rare background product. However, it seems that other people in the lab have had good results using purified and/or concentrated ligation products.
- Concentration will concentrate everything - you get more positives and more negatives. So if your problem is too few colonies this will help. If the problem is a high background, you're better off redoing the digest/CIP/extraction process. -Josh
- If you do wish to concentrate the ligation, make sure you either do phenol-chloroform extraction or use a column and not just concentrate with the speed vac.
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