All buffers should be stored at room temperature!
1. With a clean scalpel or razor blade excise the DNA fragment with care in order to minimize excess agarose. Determine the weight of the gel slice and transfer it to a clean tube.
2. For each 100 mg of agarose gel add 300 Ál buffer NT1. For highly concentrated agarose gels, add an additional 300 Ál for each percent over 1%. (For example, for a 2% gel use 600 Ál, for a 3% gel use 900 Ál, and so on.)
3. Vortex the NucleoTrap suspension thoroughly until a homogenous mixture results. Add 10 Ál of the NucleoTrap suspension to the sample. For samples containing more than 2.5 Ág of DNA, add an additional 4 Ál of the NucleoTrap suspension per additional Ág.
4. Incubate the sample for 10 minutes at 50C. Vortex the sample briefly every 2-3 minutes.
5. Centrifuge the sample for 30 seconds at >10,000 x g and discard the supernatant.
6. Add 500 Ál of buffer NT2 to the DNA containing pellet. Vortex briefly, centrifuge the sample for 30 seconds at >10,000 x g, and remove the supernatant completely. Repeat this washing step.
7. Add 500 Ál 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.
8. Air dry the pellet for 10-15 minutes. (Do not vacuum dry because over dried pellets lead to lower recoveries.)
9. Add 20Ál (60% recovery rate) - 50Ál of 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 10 minutes at room temperature, vortexing the mixture 2-3 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 with the pelleted resin could increase the yield by ~10%.
Alternative elution step for DNA larger than 5 kb.
To increase the yields of large DNA on NucleoTrap, try using 5 Ál instead of 10 Ál of the NucleoTrap suspension. Also incubate the pellet from step 8 in elution buffer at 60C for 15 minutes prior to elution.
An alternative product Nucleobond AXü is capable of isolating plasmids up to 300 kb in size in high yield. For more information on this product contact The Nest Group, Inc.
Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.
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