DIY Spin Column Protocol

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Description

A spin-column DNA extraction method using homemade buffers and silica spin columns/plates from Epoch in the USA, costs around 20cents AUD per sample.

What you need

Spin columns
or 96 well silica plates

available from Epoch

Ethanol
Tris
EDTA
SDS
Ammonium Acetate
Proteinase K
RNAse
Clean water
96 well square deep plate (2ml) to collect waste Example
96 well round deep plate (1ml) to elute DNA into for storage Example

Protocol

It's easiest to do 200 individuals at a time in two 96 well plates. If you do this they stay balanced and you don't need to discard the flow through after each step.

96 well version

  1. Homogenise tissue in the beadmill.

  2. Mix 40ml of Lysis buffer and 400µl of Proteinase K (10mg/ml).

  3. Add 200µl of Lysis buffer/ProK mix to each sample.

  4. Incubate at 50oC for 2hrs/overnight

  5. Add 2µl RNAse (10mg/ml) to each sample

  6. Incubate at 37oC for 30mins

  7. Add 200µl Binding Buffer and 200µl Ethanol to each sample

  8. Tranfer all 600µl of Lysate Binding buffer and Ethanol to the 96 well silica plate (sitting on top of a 2ml deepwell plate)

  9. Spin on high speed for 4 minutes

  10. Dispose of flow-through and add 500µl of wash buffer

  11. Spin on high speed for 2 mins

  12. Discard flow through and add another 500µl of wash buffer

  13. Spin on high speed for 15 mins to make sure membrane is dry

  14. Platce the silica plate on the 1ml deep well plate for elution

  15. Add 100µl of elution buffer to each well and incubate @ room temperature for 2 mins

  16. Spin on high speed for 1 min

Individual columns

  1. Homogenise tissue in the beadmill.

  2. Add 200µl of Lysis buffer and 2µl ProK

  3. Incubate at 50oC for 2hrs/overnight

  4. Add 2µl RNAse (10mg/ml) to each sample

  5. Incubate at 37oC for 30mins

  6. Add 200µl Binding Buffer and 200µl Ethanol to each sample

  7. Tranfer all 600µl of Lysate Binding buffer and Ethanol to the column/collection tube

  8. Spin on high speed for 1 minutes

  9. Dispose of flow-through (tip out of collection tube) and add 500µl of wash buffer

  10. Spin on high speed for 1 mins

  11. Discard flow through and add another 500µl of wash buffer

  12. Spin on high speed for 1 min

  13. Place the spin column on a new collection tube / eppie

  14. Spin on high for 2 mins

  15. Place the spin column on a clean eppie

  16. Add 100µl of elution buffer to each column and incubate @ room temperature for 2 mins

  17. Spin on high speed for 1 min

Recipes

Lysis Buffer (1 litre)

10mM Tris 2mM EDTA 1% SDS

1M Tris-HCl pH 7.510ml
0.5M EDTA4ml
SDS (sodium dodecyl sulphate) 100ml of 10%
make up to 1L with clean H2O

Binding buffer (1 litre)

3M GuHCl 3.75M NH4Ac pH 6
Guanidine Hydrochloride573.18g
Clean H2O 500ml
7.5M Ammonium Acetate500ml
Adjust pH to 6 using glacial Acetic acid
See HERE for notes on different binding buffer options.

Wash Buffer (1 litres)

10 mM Tris-HCl pH 7.5, 80% ethanol

1M Tris-HCl pH 7.510ml
96% ethanol800ml
Clean H2O190ml

Elution Buffer (500ml)

10mM Tris-Cl, pH 8.5

1M Tris-HCl pH 8.55ml
Ultrapure DNAse and RNAse free H2O495ml


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