Smolke:Protocols/Ligation: Difference between revisions
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*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, >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. | *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 | *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 | **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 | ||
Revision as of 14:34, 15 October 2009
General setup
Reaction time
Concentration and purification after ligation
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