SILICA BASED DNA EXTRACTION by Gavin Hinten <G.N.Hinten@sheffield.ac.uk>
Based on:
Elphinstone, MS. G N. Hinten, MJ. Anderson and CJ. Nock (2003) An inexpensive and
high-throughput procedure to extract and purify total genomic DNA for population studies.
Molecular Ecology Notes 3, 317–320.
Requirements: (Already in lab)
Made up glass beads (one 2 ml vial is enough) (pour into allocated tray)
Large multi-pipette
Binding Buffer (pour into allocated tray)
Wash Solution (pour into allocated tray)
Vacuum Pump
Vacuum manifold
For 96 samples.
1 box 10 µl pipette tips and Multi-pipette
2 boxes truncated tips for pipeting 200 µl at a time + 1 row of normal 200 µl tips
Filterplates: Unifilter Whatman 800
AB gene Plates for collection
PROCEDURE:
1. Do the proteinase K digestion according to protocol – please add details. Denature
10 min at 95 ºC.
2. Aliquot 50 µl for the protein K digest into 200 + µl 96 plate wells, freeze the rest in 20 ºC
3. Warm 1 15 ml of autoclaved double distilled H2O to 70 ºC (to use in last step)
4. Empty 1 vial of Glassbeads into the tray on the shaker. Shake at low speed. Using
a multi-pipette, add 10µl of beads into each well.
5. Pour 15 ml binding Buffer to allocated tray. Add 150 µl Binding Buffer into each well
using a multi-channel pipette and truncated tips. Pipette gently up and down.
6. Transfer samples to unifilter Whatman 800 plate in vacuum manifold.
7. Turn vacuum pump on, then turn manifold on (10 inch/cm 2), let liquid go trough then
Turn manifold off first, then pump off (This will increase the life of the pump). Empty
collection tray.
8. Add 200 µl ice cold Wash Solution (you can use one row of tips if you pipette
without touching wells).
9. Turn vacuum pump on, then turn manifold on (10 inch/cm 2), let liquid go trough then
Turn manifold off first, then pump off (This will increase the life of the pump). Empty
collection tray.
10. Repeat step 8 and 9, let the pump and manifold be on for 5 min during this 2 nd
wash, to ensure that the membrane is completely dry and minimize the risk of cross
contamination between wells.
11. Place a clean AB Gene collection plate under the Unifilter tray.
12. Add 50 µl of 70 ºC (makes elution of DNA more efficient) MQ H2O to each well. Let
stand for a few minutes.
13. Turn vacuum pump on, manifold on, wait for the eluted DNA to go through, turn
manifold off, pump off.
14. Run out 4 µl of DNA with 3 µl 6x loading buffer on a 1.0% agarose gel.
15. Store the stock DNA in -20 ºC, use aliquots for your lab-work.
BUFFERS: (Already in lab)
6M NaI Binding Buffer (for 500 ml):
454 g sodium iodide
7.5 g sodium sulphite
Adjust volume to 500 ml with autoclaved double distilled H2O
Filter through filter a 0.45 µm filter
Add 3 g sodium sulphite to saturate
Store in dark at 4 degree celcius (foil covered)
New Wash Solution: (1 Litre) (standard TNE ?)
Final Concentrations Required:
20 mM Tris.HCl pH 7.4
0.1 M NaCl ??
1mM EDTA
Into sterile bottle add:
1.2 g Tris.HCl
How much/ volume? NaCl
0.186 g EDTA
make up to 500 ml with autoclaved double distilled H2O
AUTOCLAVE ABOVE 15 psi 20 minutes
When cooled add 500 ml 100% Ethanol