Procedure for the Purification of PCR Products

For a faster, less expensive technique, see NucleoSpin Extract 2 in 1 kit

The following protocol was validated for the purification of PCR products. The purified DNA fragments can be used for direct sequence analysis, cloning experiments or subsequent amplifications. Residual primers, NTP's and Taq polymerase can be separated very easily with the NUCLEOBOND AX cartridge system. The conditions described below are for the AX 5 and AX 20 cartridges.

If the DNA needs to be purified more rapidly and absolute purity is not required, then an alternative product such as NucleoSpin Extract or NucleoTraPCR should be used. The latter kits consist of a silica membrane in a spin column format or loose resin, respectively. DNA binding is performed using chaotropic salts and elution is accomplished with a low ionic strength buffer such as TE. Please refer to the NucleoSpin and NucleoTrap Properties and Applications Guide for further details on these kits.

Special conditions for the type of NUCLEOBOND AX cartridge used

Step of the procedure AX 5 AX 20 Maximum amount of dsDNA 5 ”g 20 ”g

1. Extract the reaction mix with an yes yes equal volume of chloroform to remove the organic phase (mineral oil).

2. Transfer the aqueous phase to a 1.0 ml 1.0 ml fresh tube and add buffer N2. (For products smaller than 100 bp, substitute N1 for N2 in this step. (NOTE 2)

3. Check that the pH is no greater then yes yes 6.3. If it is, titrate it as required with dilute phosphoric acid.

4. Equilibrate the cartridge with buffer 1.0 ml 1.0 ml N2. (For products smaller than 100 bp, substitute N1 for N2 in this step. (NOTE 2)

5. Load the sample onto the cartridge. yes yes

6. Wash the cartridge with buffer N3. 2 x 1.0 ml 2 x 1.0 ml (For products smaller than 100 bp, substitute N2 for N3 in this step.)

7. Elute the DNA with buffer N5 and 2 x 1.0 ml 2 x 1.0 ml combine the eluates.

8. Precipitate the DNA by adding 200 ml yes yes of 3.0 M sodium acetate, pH 4.8 and 1.75 ml of room temperature iso-propanol. (Glycogen may be added to a final concentration of 1 mg/ml to act as a carrier.) Centrifuge for 10-15 minutes at 15,000 x g to pellet the DNA.

9. Wash the pellet with 70% ethanol, yes yes air dry, and dissolve in an appropriate buffer for further manipulations.

NOTE 1: The PCR process is covered by US. patents 4,683,195 and 4,683,202 owned by Hoffman-LaRoche AG.

NOTE 2: If buffer N1 is not available, a 50% solution of N2 in 15% ethanol, pH 6.3 can be used as a substitute. The latter can be made by mixing equal volumes of N2 and 15% ethanol, pH 6.3.

Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.

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Updated 1/20/98