A method is described for the purification of PCR products using an AmiconMicrocon-PCR filter (Millipore) that efficiently removes redundant dNTP and primers. This experiment is based on the PCR Laboratory Guide (Volume 2) by Kang Seo. This experiment was derived from PCR Laboratory Guide (2nd ed.) by Kang Seed and Lijia Qu.
Operation method
PCR product purification assay
Materials and Instruments
TE or deionized water PCR products Move I. Materials For more product details, please visit Aladdin Scientific website.
Centrifuge and rotor PCR filters Microtubes
1. Buffers and solutions
TE (lOmmol/L Tris-HCl, lmmol/L EDTA, pH 7.5) or deionized water
2. nucleic acids and oligonucleotides
Nucleic acids and oligonucleotides PCR products to be sequenced
3. centrifuges and rotors
Small centrifuges with fixed inclination rotors, e.g. Eppemlorf5415C
4. Specialized equipment
AmiconMicrocon-PCR filters (Millipore) or equivalent, microvial centrifuge tubes or receiver tubes
Methods
1. For each PCR product to be purified, place a filter in the receiver tube with the reservoir upwards. 2.
Add 400ul of deionized water or TE to the reservoir, taking care not to touch the membrane when adding the product. Add 100ul of PCR product, avoiding the introduction of mineral oil. If the PCR product is less than 100ul, make up with deionized water or TE and close the lid.
2. Centrifuge the product from 1 at l000 g for 15 min in a centrifuge with a fixed inclination rotor (e.g., 3300 rpm on an Eppendorf 5415C centrifuge). Note that the union of the cap and tube faces the center of the rotor.
3. Remove the filter piece and place it in a clean collection tube with the membrane side facing up (if desired, the filter piece can always be
(if desired, the filter piece can be stored until the end of the sample analysis). Carefully add 20ul of deionized water or TE to the cartridge, being careful not to touch the membrane, then invert the cartridge and place it back into a clean collection tube with the liquid side facing down.
4. Centrifuge at 1000 g for 2 min and collect the purified fragments.