The Genomic DNA Purification Kit provides a simple and rapid method for high quality genomic DNA purification from insect, nematode, mammalian tissues. 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 typical yield of genomic DNA is 3-35 μg from 10 mg of tissue. The purified high molecular weight genomic DNA is suitable for direct use in all common molecular biology applications: PCR, restriction digestion, cloning, DNA sequencing and Southern blot analysis.
Efficient: 3-35 μg of genomic DNA from 10 mg of tissue.
Fast: Procedure takes only 30 min.
Universal: purifies genomic DNA from various sources.
Safe: No phenol extraction step.
Convenient: Purified DNA can be directly used in all molecular biology applications.
High purity: Purified DNA is ready for downstream application such as PCR, restriction digestion.
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 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. Insects, nematodes or mammalian tissue (fresh or frozen) can be processed by freezing with liquid nitrogen and grinding into powder using mortar and pestle. Add ≦10 mg of tissue powder to a 1.5 ml microcentrifuge tube.
2. Add 200 μl Solution DS. Mix immediately and thoroughly by brief 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, Mix thoroughly by brief vortexing or inverting. Incubate at 55 ?C until yield a homogeneous solution (1-3 hours or overnight).
4. add 220 μl Solution MS, Mix thoroughly by brief vortexing or inverting. Incubate at 65 ?C for 10 min (inverting several times to yield a homogeneous solution).
5. Add 220 μl ethanol (96–100%) to the lysate, and mix thoroughly by brief vortexing or inverting.
6. Pipet the mixture from step 5 into the spin column placed in a 2 ml collection tube (provided). Centrifuge at 12,000 rpm for 1 min. Discard flow-through.
note ? Genomic DNA is adsorbed on the silica membrane of the column in this step.
7. add 500 μl Wash Buffer PS, and centrifuge for 1 min at 12,000 rpm. Discard flow-through.
8. Add 500 μl Wash Buffer PE, and centrifuge for 1 min at 12,000 rpm. Discard flow-through.
Note ? Wash Buffer PE must be diluted with ethanol (96-100%) previously.
9. Repeat step 8.
10. centrifuge for 3 min at 12,000 rpm to dry the column membrane. Discard flow-through and collection tube.
note ? 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.
11. 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.
Note ? 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.
12. Centrifuge for 2 min at 12,000 rpm. The tube contains the purified DNA. Store the DNA at -20 ?C.