TITLE: Purifying DNA from Agarose with Homemade Glass Milk
AUTHORS: Joanne Kamens

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>You don't need an expensive kit to purify DNA. I used to make my own (very very cheap) glass milk from this protocol. It's hard to find online these days so I'm adding it here and will share. You can read more about this on an Addgene blog </span><a href="https://blog.addgene.org/dna-purification-without-a-kit" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://blog.addgene.org/dna-purification-without-a-kit</span></a></div></div>

[STEPS]
?. Preparaton of Glass Powder (Steps 1-4)Resuspend 400 g of glass powder in 800 ml double distilled (dd) H20 in a 2 liter flask with a stir barStir for 60 minutes
?. Collect the "fines"Remove and keep the supernatant by gentle pouring, leaving all settled glass. This contains fine particles which is what you want to collect. Pellet in centrifuge 10 minutes 6000 RPM
?. Almost boil glass fines in acid to cleanResuspend pellet in 250 ml ddH20. Add Nitric Acid to 50%Heres' the tricky part. Bring close to a boil in a fume hood (! must do in fume hood!)Allow to cool.
?. Pellet and resuspend to generate stock aliquots for storagePellet glass as before, was 6 times with ddH2O. Check pH and ensure it is neutral (same as water)Resuspend with equal volume ddH2O to the pellet volume and aliquote in 50 ul aliquots for storage in freezer so it stays suspended. It will last forever and might provide a lifetime supply.
?. Prepare NaI Binding Solution90.8 g NaI1.5 g Na2SO4in 100 ml ddH2O. Let dissolve but might have a few particles.Filter through Whatman No. 1 paper (recommend into a dark bottle)Put dialysis bag containing 0.5g Na2SO4 in bottle to keep the solution saturated.Wrap bottle in foil and store at 4C. Periodically check for discoloration. If the solution turns brown it's time for new solution. (I've never tested how it works brown, it might)Let dissolve but might have a few particles.Filter through Whatman No. 1 paper (recommend into a dark bottle)Put dialysis bag containing 0.5g Na2SO4 in bottle to keep the solution saturated.Wrap bottle in foil and store at 4C. Periodically check for discoloration. If the solution turns brown it's time for new solution. (I've never tested how it works brown, it might)
?. Prepare Glass Milk Wash solution form stocks100 mM NaCl1 mM EDTA50% EtOH10 mM Tris pH 7.5Protocols recommend storage at -20C but I stored at 4C with no apparent ill effects.
?. DNA Purificaiton (Steps 7 - 12)DNA binds to glass at high salt and low temperature, elutes at low salt and higher temperature.Get to know the weight of your gel slices by weighing a few.  0.1, 0.5 and 1 gram for example. Once you know, you'll be able to know on sight without weighing.Add 2  ml NaI solution per gram of gel. This does not need to be exact.
?. Dissolve gelIncubate at 37 C, mixing occasionally until agarose is totally dissolved. Might take longer for TBE gels. Can also do this in warmer bath to 50 C to speed this up.
?. Wash Glass with DNA boundSpin suspension 5 seconds in centrifuge, remove and discard supernatant by taking off with a micro pipet.Wash pellet by resuspending in 250 ul NaI solution (or 10X volume of glass if larger)Spin, resuspend to wash pellet 2-3 times with EtOH Glass Milk Wash solution  (~ same volume). Taking off supernatant with a micro pipet gently.
?. Remove and keep eluted DNA in supernatantSpin 1 min in microfuge and remove eluted DNA in supernatant.DNA is ready for ligation, restriction, radiolabelling etc.
?. Dry Pellet and Elute DNADry pellet well, removing all residual liquid (air dry for a short time and/or  carefully use KimwipeResuspend pellet in ddH2O or TE (> 10 microliter) and elute DNA at 37 C for >5-10 mins.
?. If you want to purify DNA that is already in solution (not in a gel) you can add 3 volumes of NaI solution, immediatelyadd glass and put on ice. Follow step 10-12 to wash and elute.
?. Bind to GlassAdd 1 microliter of glass slurry per microgram of DNA. For smaller amounts, I used about 1 ul regardless, sometimes keeping a more diluted slurry for smaller amounts and so as not to waste the reagent. Incubate DNA/glass on ice 5  mins, mixing occasionally