Problems with SalI digestion - (Aug/04/2006 )

My lab has been using SalI and has been having a lot of problems with it. I want to redesign our system but I want to use sites that are reliably cut from PCR products. I already know we need some overhang beyond the restriction site, but for some reason, we've always had problems with SalI.

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salI is one of the enzymes i have been having problems with.
u can check NEB website to see how efficient is an enzyme working in different lenghts of overhangs beyond the restriction site. below is the link:
http://www.neb.com/nebecomm/tech_reference...ized_vector.asp

u may also look at the site below, some of the links in it might be interesting for u.

http://www.neb.com/nebecomm/tech_reference...mes/default.asp?

good luck

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I have had similar problems with SalI, but the funny part is it has worked like a charm for some vectors and real bad for others. I dont prefer it.

I am digesting with SalI after I had digested with the other enzyme first. This seems to b working better than double digest with SalI.

Neb's technical reference for enzymes is great to know abt different properties of enzymes.

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While you're on NEB, have a look at their page for SalI. Their 'Notes' and 'FAQs' sections on this enzyme have some interesting observations:

Notes
General notes:

1. Conditions of low ionic strength, high enzyme concentration, glycerol concentration > 5%, or pH > 8.0 may result in star activity.
2. GTm5CGAC is resistant to cleavage (in eukaryotes).
3. When cleaving close to the end of DNA fragments, cleavage should be done at 37°C for 1 hour using 10 units/µg of DNA with a minimum of 3 bases on each side of the recognition sequence.
4. Supercoiled forms of pBR322 and pUC require 10-fold overdigestion with SalI to achieve complete digestion.

FAQs

1. Is SalI affected by methylation?
2. Does SalI have trouble cleaving PCR products?
3. Does SalI have trouble cleaving PCR products?
4. Are more units of SalI required to cut supercoiled DNA than lambda DNA?
5. Are SalI buffer and BamHI buffer idententical (sic)?

Note that (in addition to spelling "identical" incorrectly in FAQ #5) they list "Does SalI have trouble cleaving PCR products?" twice -- it must be a very frequently asked question!

The answer to both entries is the same: "SalI has trouble cleaving PCR products."

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Sal I does not cut at all in anything but it's own unique buffer, which unfortunately doesn't work well with a lot of restriction enzymes.

I would avoid this enzyme entirely, if at all possible. It's not that hard to make it the sequence you want through pcr.

-Matt

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i've added ACGC before my salI site on the primer, and works good.

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