4 enzyme digest and 3 piece ligation? - (Nov/02/2007 )

1) Does anyone advise against using 4 enzymes simultaneously in a restriction digest as long as the enzymes constitute less than 10% of the ttl reaction volume and can be used in the same buffer? Do you think this will work?

2) I plan to perform this quadruple digest on two separate, but almost identical plasmids (there's a single mutation in each one that I want to eliminate by cutting and pasting the correct pieces together into a new vector. The product from each clone will be a degraded vector that I don't want, and two pieces of DNA (1.7kb and 2.7kb) that I want to insert into a 7kb mammalian expression vector. Should I try to ligate the two small fragments together first (the 1.7 and 2.7)? The 1.7 frag has an XhoI overhang, the 2.7 frag has a NotI overhang, and the other ends of both small pieces are a blunt cut by BsabI (which is what I want to stick together). ...then, should I gel purify this 4.4kb fragment and insert into my vector (which also has NotI and XhoI overhangs)? ...or should I just mix all three pieces of DNA together and add ligase and buffer?

Thanks!

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Yes this can be done. I would keep the enzyme volume less then 5% of the total reaction volume. Also make check your DNA sequence, you don't want any unexpected surprises if you miss an additional restriction site.



I would mix all three fragments together and ligate them in the same reaction mix. However do note this is one of the harder multiway ligations, blunt ends are involved. Which are not the easiest things to ligate. Anyway to revise the strategy and avoid a blunt end ligation?.

When conducting multiway ligation you should avoid quick ligase.

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Thank you so much for the helpful suggestions. It is good to hear "yes this can be done." I don't think the strategy can be revised. The insert only has one rest site in the correct position for my "cut and paste" needs, and it's a blunt cut site. ...as for the 3 part ligation, would you advise treating both of the small fragments as the insert? ...in other words, using them in equal proportions but both at 3X more than the 7kb vector? ...and ligating 18 or more hours with T4 ligase? Do you think I should check more than 30 preps? ...assuming I get colonies.

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Yes. Have the ratio of insert1:insert2:vector at 1:1:1 or if you desire 3:3:1. (3X more insert) This is a mol ratio. (number of molecules) And after looking at your inserts and vector sizes again. I would also strong advice that you use company made supercompetent cells. GeneHogs from strategene or equivalent. The final molecule size is around 10kb, which is the point I have notice that ligation work suddenly becomes harder.

In a similar vein, I think you should check more then 30. Better yet, consider doing a colony PCR. It will help save time and provided that you have a multichannel pipette you can easily test anywhere from 48 to 96 to 1000 colonies in a day. (almost the same level of a colony hybridisation). I would check at least 96 colonies with colony PCR.

What you need is a pair of primers that amplifies across junctions, either between InsertA and Insert B or a junction between insert and vector. Taq can only reliabily amplify fragments below 1kb without optimisation. So it is best to design the check primers such that the product is around between 800bp to 300bp. 500bp product size is kind of okay.

PCR is very sensitive, and it can pick up left over insert DNA from the ligation mix after plating the cells and growing them. Thus the importance of amplfying across junctions, as a junction between DNA fragments is unique to a ligated molecules. Using primers that amplify a DNA insert or a fragment of an insert will give false +ve results.

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