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E.Z.N.A. Cycle-Pure Kit Spin Protocol

互联网2013-11-13

2488

实验试剂

1. Optional: Sterile deionized water

2. Absolute ethanol (~ 96-100%)

实验设备

1. Microcentrifuge capable of at least 13,000x g

2. Nuclease-free 1.5ml centrifuge tubes

实验步骤

1. Perform agarose gel/ethidium bromide electrophoresis to analyze PCR product.

2. Determine the volume of the PCR reaction, transfer the sample into a clean 1.5ml microcentrifuge tube, and add 4-5 volumes of Buffer CP. For PCR products < 200bp add 6 volumes of Buffer CP.

3. Vortex thoroughly to mix. Briefly spin the tube to collect any drops from the inside of the lid.

4. Place a HiBind® DNA column in a provided 2ml collection tube. Add 100ul Equilibration Buffer to the column. Incubate at room temperature for 4 minutes. Spin at maximum speed for 20 seconds.

5. Apply the sample to the HiBind® DNA column and centrifuge at 10,000x g for 1 min at room temperature. Discard the flow-through.

6. Wash the HiBind® DNA column by adding 700ul of DNA Wash Buffer diluted with absolute ethanol and centrifuge as above.

IMPORTANT: DNA Wash Buffer must be diluted with absolute ethanol before use. Refer to label for instructions. If refrigerated, DNA Wash Buffer must be brought to room temperature before use.

7. Discard liquid and repeat Step 7 using 500ul of DNA Wash Buffer.

8. Discard liquid and centrifuge the empty HiBind® DNA column for 2 min at maxi speed (13,000x g) to dry the column matrix. This is critical for good yields.

9. Place HiBind® DNA column into a clean 1.5ml microcentrifuge tube. Add 30-50ul (depending on desired concentration of final product) of Elution Buffer (10mM Tris, pH8.5) or water directly onto the column matrix and centrifuge for 1 min at 13,000x g to elute DNA.This represents approximately 80-90% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration.

10. Yield and quality of DNA: Determine the absorbance of an appropriate dilution of the sample at 260nm and then at 280nm. The DNA concentration is calculated as follows:

260 DNA concentration = A x 50 x (Dilution Factor) ug/ml

Fragments greater than 500bp in length can routinely be purified at > 80% yield. 260 Bands ranging from 100bp to 500bp gives yields of 60%- 90%. The ratio of (A )/280 (A ) is an indication of nucleic acid purity. Alternatively, yield (as well as quality) can sometimes be best determined by agarose gel/ethidium bromide electrophoresis.

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