The kit is based on the defined size silica matrix used for purification of DNA from agarose gels.
COMPONENTS OF THE STANDARD KIT
- 15 ml MELT solution
- 1 ml BIND solution
- 3 ml 40x WASH solution
(for 120 ml WASH solution mix with 60 ml ethanol and 57 ml H2O)
COMPONENTS OF THE SAMPLE KIT
- 1 ml MELT solution
- 20 µl BIND solution
- 2 ml 1x WASH solution
- MELT solution: 6 M NaI
- BIND solution: DNA-binding silica matrix
- WASH solution: 50 mM NaCI 10 mM Tris-HCI pH 7.5; 2,5 mM EDTA; 50% v/v ethanol
Store the components of the DNA purification kit at +4ºC in the dark.
- Add water to MELT powder to obtain 15 ml solution.
- Mix 60 ml ethanol and 57 ml water with 3 ml of 40x WASH concentrate. For sample kit, mix 2 ml ethanol and 1,9 ml water with 100 µl of 40x WASH concentrate.
- Excise the DNA fragment from agarose gel and add one volume of MELT (150-300 µl).
- Melt the gel slice at 65°C for 5 min. Cool down
- Add 5-15 µl of BIND (will bind up to 4 µg DNA) and incubate 5 min at room temperature.
- Centrifuge 1 min at 7000 rpm, remove the supernatant and keep it for your reference.
- Add 500 µl of WASH solution to the pellet and vortex. Centrifuge 1 min at 7000 rpm. Wash twice.
- Dry pellet at room temperature. Speedvac and air-drying are admissible.
- Elute DNA by resuspending the pellet in 50-100 µl water and incubate 5 min at 55°C. Centrifuge 5 min at max. speed.
- Transfer the supernatant in another tube. If necessary, repeat centrifugation step to clean your sample from the remaining BIND. Keep this DNA probe at +4°C or -20°C. If necessary concentrate or dry DNA in Speedvac.
The DNA purified by this protocol can be used for digestion with restriction enzymes, for ligation, transformation and labelling with Klenow enzyme. This kit can also be used for purification of PCR products. DNA fragments from 20 bp up to 10 kb length can be successfully purified with this kit.