The Genomic DNA system uses the silica-gel-membrane technology for simple and fast isolation of Genomic DNA without phenol/chloroform. Homogenization is not necessary since tissues are directly lysed by Proteinase K. The buffer system is optimized to allow selective binding of DNA to the silica-gel membrane. The simple centrifugation protocol completely removes contaminants such as proteins, divalent cations, and secondary metabolites. Pure DNA is then eluted in water or low-salt buffer, ready to use. The kit is suitable for anticoagulated blood. 400 μl whole blood yield 3-10 μg high pure genomic DNA. The purified DNA can be used in common downstream applications such as sequencing, restriction digestion,etc.
High yield: 400 μl whole blood yield 3-10 μg genomic DNA
High purity: Purified DNA is ready for downstream application such as PCR, restriction digestion.
Safe: No phenol/chloroform extraction required.
Purified DNA is free from contaminants and enzyme inhibitors, and typically has A260/A280 ratios between 1.7 and 1.9, and is suitable for applications such as:
ü Restriction digestion
ü Southern blotting
ü Library construction
Store Protein K at -20℃, other reagents can be stored at room temperature for up to 1 year. Any precipitate in the Solution DS and Solution MS can be re-dissolved by incubating at 37°C before use.
After the ethanol has been added, mark the check box on the bottle to indicate the completed step.
? Examine the solution for precipitates before each use. Re-dissolve any precipitate by warming the solution to 37°C and cooling to 25°C.
? All purification steps should be carried out at room temperature.
1. Add 400 μl anticoagulated blood into a microcentrifuge tube (if using <400 μl blood sample, adjust the sample volume to 400 μl using PBS), add 800μl Solution RS to the tube. Mix thoroughly by brief vortexing or inverting. Centrifuge for 3 min at 5,000 rpm, discard the supernatant.
? The pellet is white or pink. if the pellet’s colour is dark red, it indicates that the lysis process is not thorough, you may add another 500 μl Solution RS to lyse the sample.
? Blood from mammals contains erythrocytes without nuclei. Blood from animals such as birds, fishes, or frogs (amphibians) contains nucleated erythrocytes. For mammals, 400 μl blood can yield 3-10 μg genomic DNA. For blood with nucleated erythrocytes, the volume of blood should not exceed 20 μl per tube and can yield ~40 μg genomic DNA.
2. Add 200 μl Solution DS. Mix immediately and thoroughly bybrief vortexing or inverting.
Optional ? If RNA-free genomic DNA is required, add 4 μl RNase A (100 mg/ml) and incubate for 5 min at room temperature. RNase A can be purchased separately.
3. add 20 μl Proteinase K and 220 μl Solution MS, Mix thoroughly bybrief vortexing or inverting. Incubate at 65 ?C for 10 min (inverting several times to yield a homogeneous solution).
4. Add 220 μl ethanol (96–100%) to the lysate, and mix thoroughly by brief vortexing or inverting.
5. Pipet the mixture from step 4 into the spin column placed in a 2 ml collection tube (provided). Centrifuge at 12,000 rpm for 1 min. Discard flow-through.
? Genomic DNA is adsorbed on the silica membrane of the column in this step.
6. add 500 μl Wash Buffer PS, and centrifuge for 1 min at 12,000 rpm. Discard flow-through.
7. Add 500 μl Wash Buffer PE, and centrifuge for 1 min at 12,000 rpm. Discard flow-through.
? Wash Buffer PE must be diluted with ethanol (96-100%) previously.
8. Repeat step 7.
9. centrifuge for 3 min at 12,000 rpm to dry the column membrane. Discard flow-through and collection tube.
? Since residual ethanol may interfere with subsequent reactions, it is important to dry the membrane of the spin column. This centrifugation step ensures that no residual ethanol will be carried during the following elution step. If carryover of ethanol occurs, empty the collection tube, then reuse it after centrifuging for 1 min at 12,000 rpm.
10. Place the spin column in a clean 1.5 ml microcentrifuge tube (not provided), and pipet 30-100 μl Elution Buffer TE directly onto the membrane. Incubate at room temperature for 2 min.
? Elution buffer TE can be replaced by deionized water. But the pH should be 8.0-8.5.
? Prewarm Elution Buffer TE to 65 ?C can increase the yield of genomic DNA.
11. Centrifuge for 2 min at 12,000 rpm. The tube contains the purified DNA. Store the DNA at -20 ?C.