Will restrictases cut this close? - (Aug/16/2007 )

Hi.

I want to digest my vector's MCS with BamHI and SalI, which have cutting sites right next to each other: ... GGATCC GTCGAC... (first six letters represent BamHI site, next six - SalI).
Will they cut so close without interferance, or is it too close for double digest? Thank you for your suggestions.

---

I don't know, but there is a quick way to find out.

Digest your vector with one of the enzymes, and separately with both of the enzymes.

Clean the reactions (on a column, or a size exclusion plate, or something similar. You should be left with just the linearized vector DNA, and none of the oligos that might be generated in the double digest.)

Add a little ligation buffer and ligase, and ligate for 15 minutes. Transform into competent cells.

The single digest should give you tons and tons of colonies. The double digest will give you tons and tons of colonies ONLY if the vector could only be cut by one enzyme. If both enzymes were able to cut, then you will not get nearly as many colonies in your ligation/transformation.

You can get your answer to this by tomorrow morning. The actual benchwork is <2 hours. Make sure you use the same DNA concentration in both tubes so that you can compare the numbers directly. I would use about 100ng in the digest, and I would ligate about 10 ng in about 100 microliters (to keep the concentration low to promote self-ligation.)

---

I am looking at the New England Biolabs catalogue (they are one of the better sources for info regarding the various Restriction enzymes) and that is likely going to be a difficult digest IME.

First, the cut sites are very close to each other... some enzymes require a minimum number of free neucleotides adjacent to their cut site to function efficiently. Because you have no insert to be removed, it will be nearly impossible to determine if both cut sites have been digested or if only one of them has been opened. If you go to ligate a new insert into there you may only reclose your linerized vector. You would not know this had happened until after a few days of ligating, transforming, mini-prepping, redigesting etc.

Second, these two enzymes have different buffer requirements. Under the NEB system SalI requires Buffer #3 but BamHI only has 50% efficiency in Buffer #3. Also, note that both of these enzymes exhibit star activity. You might be able to digest SalI first, then BamHI after you remove the old buffer#3 but you will likely be losing DNA between the two reactions.

My advice would be to find any other digestion options. If there are none, digest one, clean the DNA to remove buffer + enzyme by whatever method you prefer, then do the second digest. Just be sure to start with more DNA than you would have otherwise.

Good luck!

---

Nope, it don't work.
Again refering NEB technical guide, there are not enough bp around BamHI to cut efficiency. (Even if you changed buffers) (And assuming SalI was used first)

Also I am prejudice against using SalI. In my experience DNA cut by SalI do not ligate well. THe DNA cuts okay enough but come ligation, it just does not ligate.

Try something else. Do you have any other restriction site you can use?

---