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One of Many Ways to Use Rose DEAE-Filled MicroColumns™ for Purifying PCR Products from Amplifications, DNA from Enzyme Reactions, or Plasmid DNA from Bacterial Lysates
1. Prepare MicroColumn for Use
a) Compound a high salt buffer solution of TE (Tris-EDTA) (pH 7.6) containing 0.6 M NaCl.
b) Clean the DEAE resin by pipetting 200ul of this TE high salt buffer solution into a BioPak MicroColumn. Dispense and discard solution. Repeat 1x.
c) Wash away residual salt by pipetting 200ul of TE buffer (pH 7.6) (w/o) NaCl into the same BioPak MicroColumn. Dispense and discard solution. Repeat 1x.
d) Compound a low salt buffer solution of TE (pH 7.6) containing 0.1 M NaCl.
e) Complete preparation of the DEAE resin by pipetting 200ul of this TE low salt buffer solution into the same BioPak MicroColumn. Dispense and discard solution. Repeat 1x.
- To maintain hydration of resin, never completely dispense solutions from the MicroColumn.
2. Bind DNA
a) Equilibrate by bringing the ionic composition of sample to that of binding buffer solution bis-tris (pH 5.0). For details, refer to General Information pages on Buffers.
b) If volume of DNA sample is less than 50ul, bring volume up to 200ul with bris-Tris (pH 5.0). Or, if sample volume is greater than 50ul, add bis-Tris (pH 5.0) in a 3:1 volume ratio to sample.
c) Pipette 200ul of this sample solution into same BioPak MicroColumn. Pipette up and down 2x.
d) Dispense and save sample solution from step 2(c) in a clean tube or well.
- Ideal volume of sample solution is 200ul.
- If volume of sample solution (including bis-Tris) is greater than 200ul, and binding capacity of the MicroColumn is not exceeded, repeat steps 2(c) and 2(d) with any remaining sample solution.
- If quantity of DNA in the sample solution exceeds binding capacity of the MicroColumn, Wash and Elute as shown below. Then, repeat steps 1 through 4 using the same MicroColumn.
3. Wash DNA
a) Compound a mid-salt buffer solution of TE (pH 7.6) containing 0.3 M NaCl.
b) Pipette 100ul of this TE mid-salt buffer solution into the same BioPak MicroColumn. Dispense and discard soultion. Repeat 2x.
4. Elute DNA
a) Use previously compounded 0.6 M NaCl:TE high salt buffer solution (pH 7.6) from step 1(a).
b) Pipette 100ul of this TE high salt buffer solution into the same BioPak MicroColumn.
c) Pipette eluent up and down several times into an empty tube or well.
d) Dispense purified target molecule into your collecting tube or well.
- After elution, it may be desirable to extract once with phenol chloroform.
5. Precipitate DNA
a) Add 2x elution volume of 95% ethanol:ddH20, from steps 4(c) and 4(d), into same collecting tube.
b) Place sample at -70C for 10 minutes. Then, centrifuge for 15 minutes at 12,000 x g.
c) Carefully wash DNA pellet with cold 80% ethanol:ddH20. Centrifuge for 5 minutes at 12,000 x g.
6. Dry DNA
a) Recover dsDNA by evaporating eluate to dryness in a SpeedVac for 5 minutes.
7. Redissolve DNA
a) Resuspend dsDNA into 200ul of ddH20.
Protocol adapted from Molecular Cloning, A Laboratory Manual by Maniatis, Sambrook & Frish.
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