diaz@aecom.YU.EDU (Dizzy Dan) (05/11/88)
Here's this week's biochemical puzzler (you all did well on the
antisense RNA trick question):
I am interested in obtaining tritiated (or otherwise labeled)
single-stranded linear DNA of uniform length (has to be at least 1knt
long) for exonuclease assays.
M13 seems ideal since the cells will spit out labeled ssDNA (circular)
if we cook them with label. How do we make the DNA linear? Do we shear
it, or cut it with a restriction endonuclease with ss activity
(expensive!) or what? The DNA does not have to be linearized in the same
place, but it must be linear, and cut once, or at most a few times.
--
dn/dx Dept Molecular Biology diaz@aecom.yu.edu
Dizzy Dan Al Einstein's Med School Big Bad Bronx, NYnoordewi@speedy.cs.wisc.edu (Mick Noordewier) (05/12/88)
In article <1792@aecom.YU.EDU>, diaz@aecom.YU.EDU (Dizzy Dan) writes: > Here's this week's biochemical puzzler (you all did well on the > antisense RNA trick question): ^^^^^ Really? What was the trick? I thought it was an honest question! > I am interested in obtaining tritiated (or otherwise labeled) > single-stranded linear DNA of uniform length (has to be at least 1knt > long) for exonuclease assays. > > M13 seems ideal since the cells will spit out labeled ssDNA (circular) > if we cook them with label. How do we make the DNA linear? Do we shear > it, or cut it with a restriction endonuclease with ss activity > (expensive!) or what? The DNA does not have to be linearized in the same > place, but it must be linear, and cut once, or at most a few times. Restriction endonucleases with ss activity are not expensive. Enzymes known to cut ssDNA are: HhaI, HinPI and MnlI (~50% the rate of dsDNA) HaeIII (~10% the rate of dsDNA) BstNI, DdeI, HgaI, HinfI and TaqI (~100 fold lower activity than on dsDNA) AluI, HpaII, MspI, FnuDII, MboI, Sau3AI, DpnI, BbvI, FokI, HphI, MboII and SfaNI show no activity on dsDNA. You should be able to get enough with one of these enzymes at a reasonable cost. Of the above, BstNI is relatively inexpensive, and cuts M13mp18 7 times. It probably leaves at least some chunks that are a kilobase, but I don't have a map available at the moment. Since you are looking for a uniform length, you are presumably going to examine activity by looking at a gel. If a >1kb band is well enough resolved then you could simply ignore lower MW bands. Mick Noordewier noordewi@cs.wisc.edu
pell@boulder.Colorado.EDU (Anthony Pelletier) (05/13/88)
In article <5729@spool.cs.wisc.edu> noordewi@speedy.cs.wisc.edu (Mick Noordewier) writes: >In article <1792@aecom.YU.EDU>, diaz@aecom.YU.EDU (Dizzy Dan) writes: >> Here's this week's biochemical puzzler (you all did well on the >> antisense RNA trick question): > ^^^^^ >Really? What was the trick? I thought it was an honest question! > I thought it was an honest (albeit stupid) question also. I figured this dn/dx fellow as a youngish grad-student with not a whole lot of lab experience and offered a simple way to get some genuine mutants, rather then the questionable phenocopies one might get from antisense. Listen, Dan, you are not dealing with a bunch of novices here. If you think it is your place to quiz those with little experience, please mark your postings as such. Do not waste the time of other scientists. We get plenty of legitimate "puzzles" with which to deal. If your question was honest, I appologise. I would be happy to offer suggestions. But, don't try to hide the fact that you are asking for help by pretending it was quiz for us. >> I am interested in obtaining tritiated (or otherwise labeled) >> single-stranded linear DNA of uniform length (has to be at least 1knt >> long) for exonuclease assays. >> >> M13 seems ideal since the cells will spit out labeled ssDNA (circular) >> if we cook them with label. How do we make the DNA linear? Do we shear >> it, or cut it with a restriction endonuclease with ss activity >> (expensive!) or what? The DNA does not have to be linearized in the same >> place, but it must be linear, and cut once, or at most a few times. > Anyway, if the question is legitimate, consider a Polymerase Chain Reaction on an already linear fragment. You should get more than enough for exo assays (after all, if one can get a single-locus RFLP map from root cells of one hair...). If you are set on M13 and really poor, shear it and isolate linear. After all, you really don't need that much. Mazzal Tov -tony
pell@boulder.Colorado.EDU (Anthony Pelletier) (05/13/88)
In article <5729@spool.cs.wisc.edu> noordewi@speedy.cs.wisc.edu (Mick Noordewier) writes: >In article <1792@aecom.YU.EDU>, diaz@aecom.YU.EDU (Dizzy Dan) writes: >> Here's this week's biochemical puzzler (you all did well on the >> antisense RNA trick question): > ^^^^^ >Really? What was the trick? I thought it was an honest question! I thought it was an honest (albeit stupid) question also. I figured this dn/dx fellow as a youngish grad-student with not a whole lot of lab experience and offered a simple way to get some genuine mutants, rather then the questionable phenocopies one might get from antisense. Listen, Dan, you are not dealing with a bunch of novices here. If you think it is your place to quiz those with little experience, please mark your postings as such. Do not waste the time of other scientists. We get plenty of legitimate "puzzles" with which to deal. If your question was honest, I appologise. I would be happy to offer suggestions. But, don't try to hide the fact that you are asking for help by pretending it was quiz for us. >> I am interested in obtaining tritiated (or otherwise labeled) >> single-stranded linear DNA of uniform length (has to be at least 1knt >> long) for exonuclease assays. >> >> M13 seems ideal since the cells will spit out labeled ssDNA (circular) >> if we cook them with label. How do we make the DNA linear? Do we shear >> it, or cut it with a restriction endonuclease with ss activity >> (expensive!) or what? The DNA does not have to be linearized in the same >> place, but it must be linear, and cut once, or at most a few times. > Anyway, if the question is legitimate, consider a Polymerase Chain Reaction on an already linear fragment. You should get more than enough for exo assays (after all, if one can get a single-locus RFLP map from root cells of one hair...). If you are set on M13 and really poor, shear it and isolate linear. After all, you really don't need that much. Mazzal Tov