Concentration and desalination of DNA fragments with NucleoTrap and NucleoTraPCR

This protocol describes the separation of enzymes (polymerases, kinases, phosphatases etc.) and nucleotides from DNA fragments. Using this protocol, desalination of DNA can be performed as well.

ATTENTION: Before using the NucleoTrap, or 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 3 volumes of buffer NT2 to the DNA containing sample.

2. Vortex the NucleoTrap or NucleoTraPCR suspension thoroughly until a homogenous mixture results. Add 10 l of the NucleoTrap or NucleoTraPCR suspension to the sample. For samples containing more than 5 g of DNA, add an additional 2 l of the NucleoTrap or NucleoTraPCR suspension per additional g.

3. Incubate the mixture for 10 minutes at room temperature.

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

5. Add 500 l of buffer NT3 to the sample. Vortex briefly, centrifuge the sample for 30 seconds at >10,000 x g and remove the supernatant completely. Repeat this washing step.

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

7. Add 20l - 50l TE buffer (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%.

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

Back to the Nucleobond AX applications guide

Updated 1/20/98