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 or 1 x 106-107 cultrue cells. 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.
Features
Efficient: 3-35 µg of genomic DNA from 10 mg of tissue or 1 x 106-107 culture cells.
Fast: Procedure takes only 30 min.
Universal: purifies genomic DNA from various sources.
Safe: No phenol/cholroform extraction step.
High purity: Purified DNA is ready for downstream application such as PCR, restriction digestion.
Downstream Applications
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 PCR
ü Labeling
ü Library construction
Storage
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.
Important Notes
• Prior to the initial use of the kit, dilute the Wash Buffer(PE) with thanol (96-100%):
|
N1121(50preps) |
N1122(100preps) |
|
|
Wash Buffer(PE) |
15 ml |
30 ml |
|
Ethanol |
45 ml |
90 ml |
|
Total Volume |
60 ml |
120 ml |
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.
Protocol
1.Sample Process
a. For culture cells
Harvest the cells, and transfer them to a 1.5ml microcentrifuge tube. For adherent cells, trypsinize the cells before harvesting. Centrifuge at 12,000 rpm for 1 min to pellet the cells. Remove the supernatant.
b. For animal tissue
Animal tissue (either fresh or frozen at -70°C until use) tissue can be processed by freezing with liquid nitrogen and grinding into a fine powder using a mortar and pestle. Add 10mg of this tissue powder to a 1.5ml microcentrifuge tube.
2.Add 200 μl Solution DS. Vortex vigorously to resuspend cells.It is essential that the sample and Solution DS are mixed immediately and thoroughly by vortexing or pipetting to yield a homogeneous solution.
Optional If RNA-free genomic DNA is required, add 4 μl RNase A (100 mg/ml) and incubate for 5 minutes at room temperature. RNase A (100 mg/ml) can be purchased separately .
3.Add 20 μl Proteinase K and 220 μl Solution MS. Mix thoroughly by vortexing. Incubate at 65°C for 10 minutes to yield a homogeneous solution.
4.Add 220 μl ethanol (96–100%) to the sample, and mix thoroughly by vortexing. It is important that the sample and the ethanol are mixed thoroughly. A precipitate may appear. Pipet the mixture from step 4 into the spin column placed in a 2 ml collection tube (provided). Centrifuge at 12,000rpm for 1 min. Discard flow-through.
5.Add 500 μl Wash Buffer PS, and centrifuge for 1 min at 12,000 rpm. Discard flow-through.
6.Add 500 μl Wash Buffer PE, and centrifuge for 1 min at 12,000 rpm. Discard flow-through. Repeat step 6 again.
7.centrifuge for 3 min at 12,000 rpm to dry the column membrane. Discard flow-through and collection tube.
Note It is important to dry the membrane of the spin column, since residual ethanol may interfere with subsequent reactions. This centrifugation step ensures that no residual ethanol will be carried over during the following elution.Following the centrifugation step, remove the spin column carefully so that the column does not come into contact with the flow-through, since this will result in carryover of ethanol. If carryover of ethanol occurs, empty the collection tube, then reuse it in another centrifugation for 1 min at 12,000 rpm.
8.Place the spin column in a clean 1.5 ml microcentrifuge tube (not provided), and pipet 30-100 μl Eluent Buffer AE (prewarm to 65℃)directly onto the membrane. Incubate at room temperature for 2 minutes, and then centrifuge for 2 min at 12,000 rpm to elute. The tube contains the purified DNA. Store the DNA at -20℃.