Is a Triple Restriction Digest Possible? - (Nov/17/2006 )

Hello all,

I'm attempting to excise a gene from a vector using HindIII/SalI, and ligate in a gene from another vector that is cut out with the same restriction enzymes. It's a rare day that you get two vectors with two compatible sites for such an easy swap (No PCR )

Here's my problem, though. The gene I'm excising from the first vector is a long sequence, 3.3 kb. The size of the total vector is 6.6 kb, thus I'm going to wind up with two fragments of the same size and can't gel purify. However, I've found that the gene contains a StuI pretty much right in the middle, so I could chop the gene into two pieces and gel purify the remainder of the vector. SalI, HindIII, and StuI are all allegedly compatible in the same buffer, can I do a digest with all three at the same time? Or maybe do Stu I first and follow with HindIII and SalI?

Just curious, never heard of triple digests before, but I wouldn't mind saving a step.

Thanks!

fishboy83

you can use as many restriction enzymes in a digest provided
1- all the enzymes can work in the same buffer
2- that the total enzyme volume (hence total glycerol added) does not exceed 5%. Glycerol is inhibitive of enzyme activity

I have used as many as 3 RE in a single digest many time and can vouch that if the above conditions are met your digest shoudl be okay. But do note that not all enzymes cut as fast, so you are limited to the slowest enzymes.

PS: You may want to look up SalI again. According to NEB, SalI incompatible with either HindIII and StuI. SalI only works in Buffer3. HindIII and StuI can both use buffer 2. I would suggest cutting with Hind3 and StuI first and then add some buffer3 to make it high salt and cut with SalI. I am however very prejudist against SalI, it is a very poor enzyme.. give it a lot more time to cut

Your strategy sounds good to me as long as the enzymes are compatible in the same buffer. You can do a 3-way ligation after that and join everything together at once. That should work well with the 3 different sites. Just check that none of the ends created are compatible with one another and you should be fine.

The glycerol content should be ok. Use 0.5 uL of each enzyme just to make sure. Each enzyme is stored in a 50% glycerol solution, so 0.5 uL of each enzyme in a 50 uL reaction volume will leave a final glycerol concentration of 1.5%. Isn't keeping the glycerol concentration below 5% to avoid star activity? Or is it an inhibitor too?

Have followed the same strategy as you to purify a piece that was as long as the rest of the vector. As long as your enzymes are compatible buffer-wise, go ahead (bearing in mind the glycerol concentration, which is for some enzymes at least a factor in star activity).

well i think your stategy ounds good.
But may i suggest to simply do a pcr on undigested vector?
I think this would be less time consuming screening several clones...
i know the pain of cloning....

All of my restriction enzymes are from Roche, and according to their site Sal I is 100% in SuRE cut Buffer H, while Hind III and Stu I are 50-75% in Buffer H. I've done a double digest with Sal I and Eco RV (which also has 50-75% activity in Buffer H) and it worked well. If I start with 2 ug of DNA, how long should I do the digest for? I often do overnight digestions without issue, but I keep hearing how fussy Sal I can be.

fred_33,

I'm not sure I understand why I would have to screen alot of clones using this method over PCR. I plan on gel-purifying my insert and vector after digestion, and since they will have two different sticky ends the ligation should go pretty smooth, no?

well, i meant that a better strategy, i think, would be do do a PCR with very low amount of single cut plasmid. Then you'll have your pcr product at 3300 and the plasmid at 6600.
Gel purify thepcr product and digest.
You'll don't have the so-called unwanted fragment, and don't have to triple digest.