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[Home](https://www.sigmaaldrich.com/JP/en)[Polymerase Chain Reaction Applications](https://www.sigmaaldrich.com/JP/en/applications/genomics/pcr)HighPrep™ PCR Clean Up Kit and Purification System Protocol
# HighPrep™ PCR Clean Up Kit and Purification System Protocol
## PCR clean up beads and kit for post-PCR and NGS library construction
The HighPrep™ PCR Purification System uses magnetic beads-based chemistry without centrifugation or filtration in a simple 3-step procedure that includes a bind, wash, and elution step. After HighPrep™ PCR is added to the PCR reaction sample, the protocol utilizes a magnet plate (magnet stand) for processing the PCR reaction sample. During the process, contaminants and salts are washed off and pure DNA is eluted and ready to be used in subsequent applications.
## HighPrep™ PCR Protocol Materials
__HighPrep™ PCR Clean Up Kit__
- HighPrep™ PCR paramagnetic beads solution
- Store at 4 °C. DO NOT FREEZE. HighPrep™ PCR is stable for 12 months when stored at 4 °C.
- Thoroughly shake the HighPrep™ PCR reagent to resuspend the beads before use.
__Equipment and reagents to be supplied by user:__
- 80% ethanol (Prepared from non-denatured ethanol)
- 10mM TRIS-HCL pH 8.0 (DNA Elution)
- Reagent grade water
- 1mM EDTA
__Reaction Plate:__
- For 96-well format, use a 96-well cycling plate
- For 384-well format, use a 384-well cycling plate
__Product Specifications__
Cat. No.Product DescriptionNo. of ReactionsStorage Conditions
AC-60005HighPrep™ PCR (5 mL)2784-8 °C
DO NOT FREEZE
AC-60050HighPrep™ PCR (50 mL)2,776
AC-60250HighPrep™ PCR (250 mL)13,890
AC-60500HighPrep™ PCR (500 mL)27,780
Number of reactions is based on typical 10µL PCR reaction volume. Volume of HighPrep™ PCR reagent per reaction = 1.8 x (PCR Reaction Volume)
## HighPrep™ PCR 96-Well Protocol
Be sure to bring the HighPrep™ PCR reagent to room temperature for at least 30 min before use.
1. Shake the HighPrep™ PCR reagent thoroughly to fully resuspend the magnetic beads.
2. Transfer PCR reaction to appropriate 96-well plate.
3. For 50µl reaction, adjust volume using sterile water.
| | |
|--------------------------|------------------------------|
| PCR Reaction Volume (µL) | HighPrep™ PCR Volume at 1.8X |
| 10 | 18 |
| 20 | 36 |
| 50 | 90 |
Formula used to calculate the volume of HighPrep™ PCR reagent needed for PCR reaction: HighPrep™ PCR reagent volume per reaction = 1.8 X PCR reaction volume.
4. Add HighPrep™ PCR reagent volume according to the PCR reaction.
5. See table below to determine appropriate volume.
6. Mix the HighPrep™ PCR reagent and PCR sample thoroughly by mix pipetting up and down 6-8 times.
7. Incubate the mixture for 5 minutes at room temperature.
8. Place the sample plate on the 96 magnetic separation device for 3 minutes or until the solution clears. Beads will pull to the side of the well.
9. With the sample plate still on the magnet, remove and discard the supernatant by pipetting.
__IMPORTANT: Do not disturb the attracted beads while aspirating the supernatant.__
10. With the sample plate on the magnet, add 200 µl of 80% ethanol to each well and incubate for 30 seconds at room temperature.
11. With the plate still on the magnet, remove and discard the supernatant by pipetting.
12. Repeat steps 8-9 for a total of two 80% ethanol washes.
13. Dry the beads by incubating the plate for 10-15 minutes at room temperature with the plate still on the magnetic separation device.
__IMPORTANT: It is critical to completely remove all traces of alcohol but take caution in not over drying the beads as this will reduce the yield.__
14. Remove the sample plate from the magnetic separation device. Add 40µl of elution buffer (reagent grade water, TRIS-HCl pH 8.0 or TE buffer) to each well and pipet up and down 5 times to mix. Prewarming the elution buffer at 55°C can increase the yield.
15. Incubate for 2 minutes at room temperature.
16. Place the sample plate back on the magnetic separation device and wait 3 minute or until the magnetic beads clear from solution.
17. Transfer the eluate (cleared supernatant) to a new plate for storage or for subsequent applications.
## HighPrep™ PCR 384-Well Protocol
1. Shake the HighPrep™ PCR reagent thoroughly to fully resuspend the magnetic beads.
2. Transfer PCR reaction to appropriate 384-well plate. For 50µl reaction, adjust volume using sterile water.
3. Add HighPrep™ PCR reagent volume according to the PCR reaction
| | |
|--------------------------|------------------------------|
| PCR Reaction Volume (µL) | HighPrep™ PCR Volume at 1.8X |
| 5 | 9 |
| 7 | 12 |
| 10 | 18 |
Formula used to calculate the volume of HighPrep™ PCR reagent needed for PCR reaction: HighPrep™ reagent volume per reaction = 1.8 X PCR reaction volume.
4. See table below to determine appropriate volume.
5. Mix the HighPrep™ PCR reagent and PCR sample thoroughly by mix pipetting up and down 6-8 times.
6. Incubate the mixture for 5 minutes at room temperature.
7. Place the sample plate on the 384 magnetic separation device for 2 minute or until the solution clears. Beads will pull to the side of the well.
8. With the sample plate still on the magnet, remove and discard the supernatant by pipetting.
__IMPORTANT: Do not disturb the attracted beads while aspirating the supernatant.__
09. With the sample plate on the magnet, add 30 µl of 80% ethanol to each well and incubate for 30 seconds at room temperature.
10. With the plate still on the magnet, remove and discard the supernatant by pipetting.
11. Repeat steps 8-9 for a total of two 80% ethanol washes.
12. Dry the beads by incubating the plate for 3-5 minutes at room temperature with the plate still on the magnetic separation device.
__IMPORTANT: It is critical to completely remove all traces of alcohol but take caution in not over drying the beads as this will reduce the yield.__
13. Remove the sample plate from the magnetic separation device. Add 30µl of elution buffer (reagent grade water, TRIS-HCl pH 8.0 or TE buffer) to each well and pipet up and down 5 times to mix. Prewarming the elution buffer at 55°C can increase the yield.
14. Incubate for 2 minutes at room temperature.
15. Place the sample plate back on the magnetic separation device and wait 2 minute or until the magnetic beads clear from solution.
16. Transfer the eluate (cleared supernatant) to a new plate for storage or for subsequent applications.
## Materials
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