Smolke:Protocols/Ligation

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Contents

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 concentrate 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. I have noticed 2 main types of cloning failure. The first is that I get no colonies at all, and the effects of concentrating ligation products in this case has been described above. The second and more common situation is that I get a high background of negative colonies, such that I would have to screen through many of them to get a positive construct. In this case concentrating the ligation would also be ineffective, since one would obviously enrich both positive and negative colonies without improving the positive-to-negative ratio. This, of course, is just my personal experience. --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

  1. Ymer S. . pmid:1762931. PubMed HubMed [ymer]
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