Direct purification of amplified DNA fragments with the NucleoTraPCR extraction kit

ATTENTION: Before using the NucleoTraPCR extraction kit, add ethanol to buffer NT3 (volume is given on the bottle).

All buffers should be stored at room temperature!

1. Add 1 volume of chloroform to the PCR* reaction and mix briefly. Centrifuge for 1-2 minutes at 10000 x g and room temperature and transfer the upper aqueous phase to a clean tube.

2. Vortex the NucleoTraPCR suspension thoroughly until a homogenous mixture results.

3. Adjust the volume of the reaction mixture to 100 µl with TE, pH 8.0. Add 400 µl of buffer NT2 and 10µl of the NucleoTraPCR suspension to the reaction mixture and incubate the mixture for 10 minutes at room temperature, vortexing briefly every 2 to 3 minutes.

4. Centrifuge the sample for 30 seconds at >10,000 x g and discard the supernatant.

5. Add 400 µl of buffer NT2 to the DNA containing pellet. Vortex briefly and centrifuge the sample for 30 seconds at >10,000 x g. Remove the supernatant completely.

6. Add 400 µl of buffer NT3 to the sample. Vortex briefly and centrifuge the sample for 30 seconds at >10,000 x g and remove the supernatant completely. Repeat this washing step.

7. Air dry the pellet for 15 minutes at room temperature or 37C. (Do not vacuum dry because over dried pellets lead to lower recoveries.)

8. Add 20µl (60% recovery) - 50µl TE, pH 8.5 or another low salt media (e.g. sterile water, pH 8-8.5) to the sample. Resuspend the pellet by vortexing and incubate the mixture for 5 minutes at 50C or 10 minutes at room temperature, vortexing the mixture 2 to 3 times during this elution. Centrifuge the sample for 30 seconds at >10,000 x g and transfer the DNA containing supernatant to a clean tube. Repeating this step will increase the yield by approximately 10 - 15%.

REMARKS: If the volume of the PCR* reaction mixture is >100 µl, the volumes of the buffers NT2, NT3 and the NucleoTraPCR suspension must be increased proportionally. For example, 150 µl reaction mixture would need 600 µl each of buffers NT2 and NT3 and 15 µl NucleoTraPCR suspension.

* The polymerase chain reaction (PCR) process is covered by U.S. Patent No. 4,683,105 and 4,683,202 issued to Hoffman-La Roche. Patents pending in other countries.

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

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