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 three constructs. One of the three 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[1]. -Josh I actually never heat inactivate the ligation before transformation, but this is not a bad idea if you plan to keep the ligation product for potential repeats in transformation. --YC References Ymer S. Heat inactivation of DNA ligase prior to electroporation increases transformation efficiency. Nucleic Acids Res. 1991 Dec 25;19(24):6960. DOI:10.1093/nar/19.24.6960 |