Feb 11, 2020
Purifying DNA from Agarose with Homemade Glass Milk
- 1Addgene
Protocol Citation: Joanne Kamens 2020. Purifying DNA from Agarose with Homemade Glass Milk. protocols.io https://dx.doi.org/10.17504/protocols.io.bcefitbn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
I used this protocol for 20 years and it worked almost every time. I made 2 batches of glass milk in that time. It's cheap and easy.
Created: February 11, 2020
Last Modified: February 11, 2020
Protocol Integer ID: 32935
Keywords: DNA, purification, cheap, budget ,
Abstract
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 https://blog.addgene.org/dna-purification-without-a-kit
Guidelines
This method is a cheap, effective, easy way to purify DNA from bands in an Agarose gel. The DNA will be clean and can be used in almost any procedure (all that I've tried). Note that it is rumored to only work with TAE gels, but this is not correct. It works fine with TBE with very little loss. The agarose melting step takes a bit longer and might take a bit more volume but it works just fine.
Materials
MATERIALS
Sodium iodideP212121
Sodium sulfateP212121
#1 Whatman paper Coorstek Catalog #1001-150
Nitric acid
NaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #53014
TrisBio Basic Inc.Catalog #TB0196.SIZE.2.5Kg
100% EtOHGold Shield DistributorsCatalog #DSP-CA-151
EDTAFisher ScientificCatalog #16 004Y
Silica 325 Mesh
Silica 325 mesh is a flint glass powder available from ceramic shops
Safety warnings
Bringing Nitric Acid to an almost boil can be dangerous. Use a hood, safety equipment and don't ask a lab newbie to do this step.
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 bar
Stir 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 clean
Resuspend 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 storage
Pellet 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 Solution
- 90.8 g NaI
- 1.5 g Na2SO4
- in 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)
Prepare Glass Milk Wash solution form stocks
- 100 mM NaCl
- 1 mM EDTA
- 50% EtOH
- 10 mM Tris pH 7.5
Protocols 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 gel
Incubate 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.
Bind to Glass
Add 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
Wash Glass with DNA bound
- Spin 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.
Dry Pellet and Elute DNA
Dry pellet well, removing all residual liquid (air dry for a short time and/or carefully use Kimwipe
Resuspend pellet in ddH2O or TE (> 10 microliter) and elute DNA at 37 C for >5-10 mins.
Remove and keep eluted DNA in supernatant
Spin 1 min in microfuge and remove eluted DNA in supernatant.
DNA is ready for ligation, restriction, radiolabelling etc.
If you want to purify DNA that is already in solution (not in a gel) you can add 3 volumes of NaI solution, immediately
add glass and put on ice. Follow step 10-12 to wash and elute.