14 PCR cleanup kit

E.Z.N.A.® Cycle Pure Kit Centrifugation Protocol (PCR cleanup kit)

Materials and Equipment to be Supplied by User:

•    Microcentrifuge capable of at least 13,000 x g
•    Nuclease-free 1.5 mL microcentrifuge tubes
•    100% ethanol (used to dilute wash buffer before beginning protocol)
•    Sterile, deionized water.

1. Perform agarose gel/ethidium bromide electrophoresis to analyze PCR product.
2. Determine the volume of your PCR reaction.
3. Transfer the sample into a clean 1.5 mL microcentrifuge tube (not provided).
4. Add 5 volumes CP Buffer. Record the volume you used!

Note: Volume refers to the size of your PCR reaction. For example, if your PCR reaction is 100 μL, use 500 μL CP Buffer.

5. Mix thoroughly.
6. Insert a HiBind® DNA Mini Column into a 2 mL Collection Tube (provided).
7. Add the sample from Step 5 to the HiBind® DNA Mini Column.
8. Centrifuge at maximum speed (≥13,000 x g) for 1 minute at room temperature.
9. Discard the filtrate and reuse collection tube.
10. Add 700 μL DNA Wash Buffer (make sure ethanol has already been added to buffer)
11. Centrifuge at maximum speed for 1 minute.
12. Discard the filtrate and reuse the collection tube.
13. Repeat Steps 10-12 for a second DNA Wash Buffer wash step.
14. Centrifuge the empty HiBind® DNA Mini Column at maximum speed for 2 minutes to dry the column. Note: This step is critical for removal of trace ethanol that may interfere with downstream applications.
15. Transfer the HiBind® DNA Mini Column into a clean 1.5 mL microcentrifuge tube (not provided).
16. Add 30 μL sterile deionized water directly to the center of column matrix.
17. Let sit at room temperature for 2 minutes.
18. Centrifuge at maximum speed for 1 minute to elute your DNA.
19. Store DNA at -20 °C

Note: This represents approximately 80-90% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.