Cfrench:DNAPurification1

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(New page: ==4. Purifying a PCR product from solution== If your PCR product looks pure enough to continue with (ie a single band of the expected size, with no extra unwanted DNA bands), you can puri...)
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===Materials required===
===Materials required===
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'''6 M sodium iodide.''' Weigh out 18 g of sodium iodide and add water to give a volume of 20 ml. Not very much water is required. The sodium iodide may take some time to dissolve. '''Safety note: sodium iodide was formerly listed as ‘highly toxic’. It has now been reclassified as ‘harmful’, but treat it with caution. Wear gloves.''' The solution should be stored at 4˚C. It will gradually turn yellow over time, but this does not seem to have any adverse effect on the procedure.
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'''6 M sodium iodide.''' Weigh out 18 g of sodium iodide and add water to give a volume of 20 ml. Not very much water is required (about 15 ml in my experience). The sodium iodide may take some time to dissolve. '''Safety note: sodium iodide was formerly listed as ‘highly toxic’. It has now been reclassified as ‘harmful’, but treat it with caution. Wear gloves.''' The solution should be stored at 4˚C. It will gradually turn yellow over time, but this does not seem to have any adverse effect on the procedure.
'''Glass bead suspension.''' Weight out 1 g of silica beads (eg Sigma S-5631, mixture of particle sizes from 0.5 to 10 micrometres). '''Safety note: silica powder is irritating to the eyes and lungs. Wear eye protection and a dust mask when working with dry silica powder.''' To remove the finest particles, suspend the beads in 10 ml PBS (phosphate buffered saline; I use 10 mM phosphate buffer, pH 7, with 0.9% w/v NaCl, but this is probably not critical). Allow the beads to settle for 2 hours. Remove and discard the supernatant, which will contain the finest particles, slowest to settle. Repeat this procedure. Then transfer the remaining suspension to a microcentrifuge tube, spin briefly at low speed, remove the supernatant, and resuspend the beads in 5 ml of 3 M sodium iodide: '''See safety note above re sodium iodide.''' The suspension should be stored at 4˚C. The glass beads must be vigorously resuspended before use.
'''Glass bead suspension.''' Weight out 1 g of silica beads (eg Sigma S-5631, mixture of particle sizes from 0.5 to 10 micrometres). '''Safety note: silica powder is irritating to the eyes and lungs. Wear eye protection and a dust mask when working with dry silica powder.''' To remove the finest particles, suspend the beads in 10 ml PBS (phosphate buffered saline; I use 10 mM phosphate buffer, pH 7, with 0.9% w/v NaCl, but this is probably not critical). Allow the beads to settle for 2 hours. Remove and discard the supernatant, which will contain the finest particles, slowest to settle. Repeat this procedure. Then transfer the remaining suspension to a microcentrifuge tube, spin briefly at low speed, remove the supernatant, and resuspend the beads in 5 ml of 3 M sodium iodide: '''See safety note above re sodium iodide.''' The suspension should be stored at 4˚C. The glass beads must be vigorously resuspended before use.

Revision as of 04:47, 1 August 2011

4. Purifying a PCR product from solution

If your PCR product looks pure enough to continue with (ie a single band of the expected size, with no extra unwanted DNA bands), you can purify it directly from the remaining 45 microlitres of the reaction mixture as follows.

Materials required

6 M sodium iodide. Weigh out 18 g of sodium iodide and add water to give a volume of 20 ml. Not very much water is required (about 15 ml in my experience). The sodium iodide may take some time to dissolve. Safety note: sodium iodide was formerly listed as ‘highly toxic’. It has now been reclassified as ‘harmful’, but treat it with caution. Wear gloves. The solution should be stored at 4˚C. It will gradually turn yellow over time, but this does not seem to have any adverse effect on the procedure.

Glass bead suspension. Weight out 1 g of silica beads (eg Sigma S-5631, mixture of particle sizes from 0.5 to 10 micrometres). Safety note: silica powder is irritating to the eyes and lungs. Wear eye protection and a dust mask when working with dry silica powder. To remove the finest particles, suspend the beads in 10 ml PBS (phosphate buffered saline; I use 10 mM phosphate buffer, pH 7, with 0.9% w/v NaCl, but this is probably not critical). Allow the beads to settle for 2 hours. Remove and discard the supernatant, which will contain the finest particles, slowest to settle. Repeat this procedure. Then transfer the remaining suspension to a microcentrifuge tube, spin briefly at low speed, remove the supernatant, and resuspend the beads in 5 ml of 3 M sodium iodide: See safety note above re sodium iodide. The suspension should be stored at 4˚C. The glass beads must be vigorously resuspended before use.

Wash Buffer: 10 mM Tris/HCl, pH 7.5, 2.5 mM EDTA, 50 mM NaCl, 50% v/v ethanol. To make 100 ml, weight out:

  • Tris base: 24 mg
  • Tris.HCl: 127 mg
  • NaCl: 293 mg
  • Na2EDTA: 93 mg

Dissolve in 50 ml water. Add 50 ml absolute ethanol. Store at -20˚C (it will not freeze due to the high alcohol content). This solution is stable at room temperature but must be ice-cold when used.

EB (Elution Buffer): This is 10 mM Tris, pH 8. To make 100 ml of EB, dissolve 53 mg Tris base and 88 mg Tris hydrochloride in 100 ml water. Sterilise by autoclaving.

Procedure

  • 0. Turn on a waterbath to 55˚C so that it has time to warm up before you need it in step 11.
  • 1. Transfer the 45 microlitres of PCR reaction mixture from the 0.5 ml PCR tube to a 1.5 ml microcentrifuge tube.
  • 2. Add 150 microlitres of 6 M sodium iodide.
  • 3. Add 20 microlitres of glass bead suspension. Note: mix the suspension well before removing the glass beads.
  • 4. Mix and incubate on ice for 15 minutes (or longer; the exact time is not important). If all goes well, the DNA will stick to the glass beads.
  • 5. Spin the tube briefly in a microcentrifuge to get the beads to the bottom of the tube. The speed is not critical, and 30 seonds or so should be plenty, since glass beads settle very well.
  • 6. Remove the supernatant to a waste beaker. Ultimately this can be discarded to the drains.
  • 7. Add 250 microlitres of ice-cold wash buffer. (This is stored in the freezer and should be kept on ice when out of the freezer). Mix by inversion. Do not attempt to resuspend the glass beads. Spin the tube briefly and remove the supernatant to your waste beaker. Remove as much of the supernatant as possible. The purpose of this step is to wash away sodium iodide.
  • 8. Repeat step seven twice more.
  • 9. If there is still some liquid stuck to the sides of the tube, spin again briefly and remove it. Make sure that you have removed as much of the liquid as you possibly can.
  • 10. Add 40 microlitres of EB (elution buffer). Resuspend the glass beads by pipetting up and down. Make sure that they are well resuspended.
  • 11. Incubate in a 55˚C waterbath for 10 minutes or so, mixing again by flicking the tube at the 5 minute mark (this is probably not essential, but the beads do tend to settle very quickly).
  • 12. Spin at high speed for 1 minute.
  • 13. Transfer the supernatant to a clean labelled tube. Try to avoid getting any glass beads in it. Discard the tube with the glass beads.
  • 14. The DNA should be stored at -20˚C. You can check that the purification procedure has worked properly by running another 5 microlitres of the DNA on a gel, as before.

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