The Quick Blood Genomic DNA Purification Kit provides a simple and rapid method for high quality genomic DNA purification from whole blood. Compare to traditional blood genomic DNA extraction kit, the quick system provides easier method in that erythrocytes do not need to be removed from the whole blood. The Quick Blood 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 5 µg from 200 μl of whole blood. 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
Fast: Procedure takes only 30 min.
Simple: No need to remove erythrocyte from the whole blood
Efficient: 5 µg from 200 μl of whole blood.
Safe: No phenol 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
Kit Content: (for a 50-prep kit size)
|
Component |
Volume or Size |
|
Solution DS |
15 ml |
|
Solution QS |
20 ml |
|
Proteinase K |
1 ml |
|
Wash Buffer (PS) |
30 ml |
|
Wash Buffer (PE) |
15 ml |
|
Elution Buffer TE |
5 ml |
|
Spin Columns |
50 each |
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 QS 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 ethanol (96-100%):
|
50 prep Kit |
100 prep Kit |
|
|
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.Add 200 μl blood sample to a 1.5 ml microcentrifuge tube, if the volume of blood is less than 200 μl, please add solution DS to 200 μl.
Optional If RNA-free genomic DNA is required, add 4 μl RNase A (100 mg/ml), mix thoroughly by vortexing. Incubate for 5 minutes at room temperature. RNase A (100 mg/ml) can be purchased separately .
Note ·Blood must be collected in EDTA, heparin or citrate anticoagulant tubes to prevent clotting.
·Blood from mammals contains nonnucleated erythrocytes. Blood from animals such as birds, fish, or frogs contains nucleated erythrocytes. For blood with nonnucleated erythrocytes, 200μl blood can yield 1.5-5μg genomic DNA. For blood with nucleated erythrocytes, the volume of blood do not exceed 20 μl per tube. 20 μl whole blood can yield 40 μg genomic DNA.
2.Add 20 μl Proteinase K and 220 μl Solution QS, Mix well. Incubate at 65°C for 10–15 minutes to yield a black homogeneous solution. Spin down the water beads on the wall of the tube.
3.Add 220 μl ethanol (96–100%) to the sample, and mix thoroughly by inverting. It is important that the sample and the ethanol are mixed thoroughly. A precipitate may appear. Pipet the mixture from step 3 into the spin column placed in a 2 ml collection tube (provided). Centrifuge at 12,000rpm for 1 min. Discard flow-through.
4.Add 500 μl Wash Buffer PS, and centrifuge for 1 min at 12,000 rpm. Discard flow-through.
5.Add 500 μl Wash Buffer PE, and centrifuge for 1 min at 12,000 rpm. Discard flow-through. Repeat step 6 again.
6.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.
7.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℃。
