Original Repository: Alan Mason Chesney Medical Archives. Daniel Nathans Collection
Reproduced with permission of Joanne Nathans.
Medical Subject Headings (MeSH):
DNA Restriction Enzymes
Restriction Enzymes and the "New Genetics," 1970-1980
Letter from David C. Ward to Daniel Nathans (March 16, 1971)
February 22, 1971
Enclosed are 3 tubes of E. coli B restriction endonuclease in 50 percent glycerol. The enzyme proved stable in 50 percent
glycerol at -50 degrees for at least 10 days. This preparation is the G-200 Sephadex faction as described by Roulland-Dussoix
and Boyer in BBA 195, 219, 1969; prior to glycerol addition it was in .01 KPO4, pH 7 -- .005 M mercaptoethanol -- .001 M EDTA.
Tested against high specific activity, sonicated 32P-T7 DNA (2 x 10 [to the fifth] cpm/[micro]gram), it had no detectable
exonuclease activity under the conditions of the B enzyme assay. To cleave SV40 DNA we use the following conditions:
SV40 DNA I - 20 [gamma]/ml: 6 [lambda]
Mix - : 4 [lambda]
Enzyme (50 percent glycerol): 10 [lambda]
36 degrees x 30 minutes
Stop reaction by adding EDTA to .015 M
Mix: SAM .06 M in .01 NH2SO4: 10 [lambda]
Tris C1 pH 7.6 1 M: 20 [lambda]
ATP .06 M: 10 [lambda]
DTT .03 M: 10 [lambda]
MgCl2 0.3 M: 10 [lambda]
Water: 40 [lambda]
With the amount of enzyme given above 0.03 [micro]grams DNA was converted to a heat denaturable form in 5 minutes. The reaction
slows down after about 15 minutes and generally I add more enzyme after 15 minutes. Recently I have found that the formation
of form III proceeds at a decreasing rate for > 30 minutes and I suggest therefore that you incubate up to 1 hour.
To assay the enzyme we incubate for 5 minutes using radioactive DNA and add 5 volumes of 1 x SSC. This is heated at 100 degrees
for 2 minutes, quickly chilled in ice and passed through an S+S B-6 filter to count denatured DNA. One unit of enzyme is the
amount that converts .01 [micro]grams SV40 DNA I to a denaturable form in 5 minutes.
I hope the enzyme works when it arrives and does what you need. Let me know if I can be of further help.