Smolke:Protocols/Ligation: Difference between revisions
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*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 | *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 | **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 | **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 concentration 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 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<cite>ymer</cite>. -Josh | *If you're not cleaning up the ligation, it should be heat inactivated (65C for 10-20 minutes) before transformation<cite>ymer</cite>. -Josh |
Revision as of 15:01, 15 October 2009
General setup
Reaction time
Concentration and purification after ligation
References |