JROSENBAUM.CURRY@BIONET-20.BIO.NET (Alice Curry) (04/12/89)
Is anyone out there familiar with the exoIII/S1 nuclease protocol of Henikoff (Methods in Enzymology 155:156)? I am currently having problems with this protocol as implemented in Promega's Eraseabase kit. Ironically, we have used this kit for two previous projects with great success, but the method, even with fresh components, isn't working anymore. The problems semm to be occuring after the exo deletions themselves. I'd appreciate hearing form anyone who has worked out problems with this system, (with or without Promega's kit), or from anyone who has had problems with the kit, particularly in recent months. --Alice Curry -------
CWILLIAMS.GIEGEL@BIONET-20.BIO.NET (David Giegel) (04/17/89)
Alice, I have used Promega's system with great success in the last half year. I have found several situations that can lead to inefficient cutting of the plasmid. The biggest factor influencing the digestion is the quality of the dsDNA that you are starting with. CsCl-purified DNA has given me the best results. I get less digestion starting from nicks in the DNA with plasmid prepared in this way. I have also found that protection of digestion with ApaI is tricky. This restriction enzyme may have some nonspecific endonuclease activity (or perhaps contaminating nicking activity) that makes DNA digested for prolonged times with it more susceptable to nonspecific ExoIII digestion. I ended up only digesting for 1.5-2 hrs. with this enzyme prior to treatment with the ExoIII. Finally, I have found that doing the ligation step overnight at 16 degrees C greatly enhances the number of transformants that are obtained. On the other side of the coin, I have a friend that is currently using the kit to make nested deletions for sequencing and is finding that many of the deletions that she isolates and sequences are not in consecutive order. She is getting more early time deletions appearing at sequences further along on the clone than I ever saw. Her kit was just purchased in the last several months and it could be a badlot of ExoIII but I have no data to state this definitively. The quality of the ExoIII greatly influences the results that are obtained. Let me caution you that I have tried Stratgene's ExoIII/Mung Bean Nuclease deletion kit very extensively with absolutely no success at all. Promega's kit is much easier to use and the results are usually very good. One last point that I should mention, I start the digestions with 10 ug of DNA instead of the 5 that is suggested in the instructions. If I can be of any further assistance, do not hestitate to drop me a note. Sincerely, Dave Giegel <CWILLIAMS.GIEGEL@BIONET-20> -------