Smolke:Protocols/Ligation

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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 a 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 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. --YC 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 My point is that, in my experience, the cloning will either work or not work, and concentrating the ligation has not made a deference. --YC 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. If you're not cleaning up the ligation, it should be heat inactivated (65C for 10-20 minutes) before transformation. -Josh