loren@wystan.bsd.uchicago.edu (02/20/91)
Can anyone recommend a source of glass beads suitable for use in DNA
purification? I cannot locate the source cited in the article by B.
Vogelstein. A specific catalogue number would help if there are dozens of
related choices. The Bio 101 GeneClean kits work so nicely, I can no
longer afford the habit. If anyone has any experience using DEAE cellulose
paper, I would appreciate their comments. Schleicher and Schuell smallest
unit sale is enough to last several years, but they only guarantee a one year
shelf life.
Thank You,
Loren Joseph
Dept. of Pathology
University of Chicago
e-mail: ah,err, not quite sure but it is something like
the University Computer is designated @midway.uchicago.edu and I log on as
'ljlj' but I connect to a miniVax at node wystan.bsd.uchicago.edu where I
sign on as LOREN. The minivax is also designated, at some level, as
@uchimvs1. From the miniVax I go back to the university newserver. cup@bones.biochem.ualberta.ca (02/21/91)
>Can anyone recommend a source of glass beads suitable for use in DNA >purification? I cannot locate the source cited in the article by B. >Vogelstein. A specific catalogue number would help if there are dozens of >related choices. The Bio 101 GeneClean kits work so nicely, I can no >longer afford the habit. >Loren Joseph >Dept. of Pathology >University of Chicago Yes, I switched to making my own glass milk. Works great with DNA >500 bp from TAE gels. I bought 2 kg of glass powder from a local pottery outfit, probably enough for 500ml of glass milk. I don't have the method on this machine but I'll try and post it in the next few days. You can make a lot of friends supplying your dept. Chris Upton use usercu11@ualtamts for E-mail, the other address may not be in service for a while.
cup@bones.biochem.ualberta.ca (02/21/91)
>Can anyone recommend a source of glass beads suitable for use in DNA >purification? Sorry, I forgot to say in the previous follow-up that the 2kg of glass powder cost me $2.50 (yes that is $2.50!!) Chris Upton >
levyd@mcclb0.med.nyu.edu (02/21/91)
In article <009447D1.0CC2B140@wystan.bsd.uchicago.edu>, loren@wystan.bsd.uchicago.edu writes: > Can anyone recommend a source of glass beads suitable for use in DNA > purification? I cannot locate the source cited in the article by B. > Vogelstein. A specific catalogue number would help if there are dozens of > related choices. The Bio 101 GeneClean kits work so nicely, I can no > longer afford the habit. If anyone has any experience using DEAE cellulose > paper, I would appreciate their comments. Schleicher and Schuell smallest > unit sale is enough to last several years, but they only guarantee a one year > shelf life. > Thank You, > > Loren Joseph > Dept. of Pathology > University of Chicago > e-mail: ah,err, not quite sure but it is something like > the University Computer is designated @midway.uchicago.edu and I log on as > 'ljlj' but I connect to a miniVax at node wystan.bsd.uchicago.edu where I > sign on as LOREN. The minivax is also designated, at some level, as > @uchimvs1. From the miniVax I go back to the university newserver. S&S NA45 paper works very well and the 1 yr shelf life guarantee is probably very conservative. We have successfully used material 2-3 yrs old, but there is a deterioration if it gets too old or if it is stored in a hot place where it gets too dried out. Glass powder can usually be found at art or ceramics supply stores. Ask for the finest glass milk. It is often used to mix with paint to make a light-reflective paint.
cup@bones.biochem.ualberta.ca (02/22/91)
In article <1991Feb21.094414.6987@mcclb0.med.nyu.edu> levyd@mcclb0.med.nyu.edu writes: >In article <009447D1.0CC2B140@wystan.bsd.uchicago.edu>, loren@wystan.bsd.uchicago.edu writes: >> Can anyone recommend a source of glass beads suitable for use in DNA >> purification? I cannot locate the source cited in the article by B. >> Vogelstein. I think my last note about glass milk didn't get to the world distribution of usenet. (just changing over from mail to usenet, I like it already). Well, I've been making my own glass milk for a couple of years, the recipe was given to me by Paula Traktman at Cornell Med School who got from..... It works great and costs peanuts. So I tend to use it for everything. Purify ds DNA for sequencing Switch restriction buffers purify vector and insert before ligation I'll tag the recipes at the end. PLEASE use the following for E-mail usercu11@mts.ucs.ualberta.ca Chris Upton PURIFICATION OF DNA BY BINDING TO GLASS POWDER Binding and Wash Solutions NaI solution: 90.8 g NaI 1.5 g Na2SO4 in 100 ml H2O. Filter through Whatman No.1. Put dialysis bag containing 0.5 g Na2SO4 in bottle to keep solution saturated. Store foil-wrapped at 4 oC. NEET Wash: 100 mM NaCl 1 mM EDTA 50 % EtOH 10 mM Tris pH 7.5 Store at -20 oC. DNA Purification: To purify DNA from agarose gel, weigh gel slice. Add 2 - 3 ml NaI solution per gram of gel. Incubate at 37-50 oC, mixing frequently until agarose is totally dissolved. Add 1 5l of glass slurry per 5g of DNA. Incubate on ice 5-10 mins, mixing occasionally. Spin 5-10 secs in microfuge, remove and discard supernatant. Wash glass pellet with 250 5l NaI (or 10 x volume of glass if larger). Spin and wash pellet 2-3 times with EtOH wash (same volume). Dry pellet well, removing all residual liquid (air dry or use Kimwipe carefully). Resuspend pellet in H2O or TE (> 10 5l) and elute DNA at 50 oC for 5-10 mins. Spin 1 min in microfuge and remove eluted DNA in supernatant. DNA is ready for ligation, restriction, radiolabelling etc. DNA bind to glass at high salt and low temp, elutes at low salt and high temp.! (To purify DNA from solution, add 3 volumes of NaI solution, immediately add glass and put on ice). PREPARATION OF GLASS POWDER Use silica 325 mesh (a powdered flint glass available from ceramic shops) Resuspend 400 g of glass powder in 800 ml ddH2O in a 2 litre flask. Stir for 60 mins. Allow to settle for 90 mins. Take the SUPERNATANT (which contains the "fines" of interest) and pellet in Sorvall (GSA rotor, 10 mins at 6000 rpm). Resuspend pellet in 200-300 ml ddH2O. Add nitric acid to 50 %. Bring close to boil in fume hood. Allow to cool. Pellet glass as before, wash pellet 4-6 times with JddH2O (check pH returns to neutral). Store final pellet as 50 % slurry in ddH2O. Store at -80 oC, working aliquot at 4 oC. Cost for 2.5 kg of glass powder is approx $3