Blood genomic DNA extraction experiment
Method principle
Special cell lysis and protein removal solutions (including proteinase K lysis) are used to obtain genomic DNA from anticoagulated whole blood.
[Kit composition, storage, stability]
Consumables: 96 deep well plate (1.6mL), 96 round well plate, 96 well DNA preparation plate, 96-well round-well silica gel sheet, BF-400 membrane.
ProteinaseK: Freeze-dried proteinase K can be stored at room temperature for 6 months. For long-term storage, please store it at 4°C; after dissolution, it can be stored at 2-8°C for 2 months. Do not store it at room temperature for long-term storage.
Buffer PK: Proteinase K solution, sealed at room temperature.
Buffer BL: Cell lysate, sealed and stored at room temperature.
Buffer W1B concentrate: washing liquid. Before use, add ethanol according to the quantity on the bottle, mix well, and store in airtight at room temperature.
Buffer W2 concentrate: Desalt solution. Before use, add ethanol according to the quantity on the bottle, mix well, and store in airtight at room temperature.
10xBuffer W2 (12x96 kit): Prepare Buffer W2.
EluentB: 7.5mM Tris-HCl, pH8.5, 0.3mM EDTA, airtight storage at room temperature.
Experiment preparation
1. When using for the first time, add absolute ethanol to Buffer W2 concentrate and Buffer W1B concentrate according to the volume specified on the reagent bottle.
2. BufferW2 (12x96 kit) preparation: add 15mL 10xBuffer W2, 135mL deionized water and 350mL ethanol to the provided 500mL empty bottle. 100% absolute ethanol or 95% ethanol can be used.
3. Dissolve proteinase K in Buffer PK according to the label on the bottle, do not vortex.
4. Prepare a 70℃ warm bath.
5. Before use, check whether there is precipitation in Buffer BL. If precipitation occurs, please heat it in a warm bath at 70°C until the precipitation is completely dissolved before use.
Experimental operation
1. Add 20ul proteinase K to each well of a 96-well round-well plate.
2. Add 200ul of anticoagulated whole blood to a 96-well round-well plate.
*If the volume of whole blood sample is less than 200ul, supplement to 200ul with PBS.
*If the sample is bird blood, the sample amount must be less than 10ul.
*To obtain RNA-free genomic DNA, add DNase-free RNaseA (20mg/mL) before adding Buffer BL in step 3.
3. Add 200ul Buffer BL (do not wet the edges of each hole, seal each hole with a 96-hole round-hole silica gel), and mix vigorously for 30 seconds.
4. Briefly centrifuge at 3000rpm to transfer the solution on the 96-well round-well silica gel sheet to the 96-well round-well plate. Start the centrifuge and stop when the speed reaches 3000rpm.
5. Incubate in an incubator or oven at 70°C for at least 10 minutes.
6. Briefly centrifuge at 3000rpm to transfer the solution on the 96-well round-well silica gel sheet to the 96-well round-well plate. Start the centrifuge and stop when the speed reaches 3000rpm.
7. Remove the 96-hole round-hole silica gel sheet, and add 200ul ethanol (96-100%) to each hole.
8. Seal each hole with a 96-hole round-hole silica gel sheet, and mix vigorously for 15 seconds. Centrifuge briefly at 3000 rpm to transfer the solution on the 96-well round-well silica gel sheet to the round-well plate. Start the centrifuge and stop when the speed reaches 3000rpm.
9. Place the 96-well DNA plate into a clean 96-well 1.6mL deep well plate. Remove the 96-well round-well silica gel sheet from the round-well plate, transfer the solution in each well to the 96-well DNA plate, and centrifuge at 6000 rpm for 4 min.
10. Discard the filtrate from the 96-well 1.6mL deep-well plate, return the 96-well DNA plate to the 96-well 1.6mL deep-well plate, add 500ul Buffer W1B to each well, and seal the 96-well DNA plate with a new BF-400 membrane , Centrifuge at 6000rpm for 4min.
* Make sure that absolute ethanol has been added to the Buffer W1B concentrate according to the specified volume on the reagent bottle.
11. Discard the filtrate from the 96-well 1.6mL deep-well plate, return the 96-well DNA plate to the 96-well 1.6mL deep-well plate, add 850ul Buffer W2 to each well, and seal the 96-well DNA with another new BF-400 membrane Plate, centrifuge at 6000rpm for 4min.
* Make sure that absolute ethanol has been added to the Buffer W2 concentrate according to the specified volume on the reagent bottle.
12. Discard the filtrate, return the 96-well DNA plate to a 96-well 1.6mL deep-well plate, add 400ul Buffer W2 to each well, seal the 96-well DNA plate with another new BF-400 membrane, and centrifuge at 6000 rpm for 4 minutes.
13. Place the 96-well DNA plate on a clean 96-well 1.6mL deep-well plate, seal the 96-well DNA plate with BF-400 membrane, and centrifuge at 6000 rpm for 15 minutes.
14. Place the 96-well DNA plate on another clean 96-well 1.6mL deep-well plate, add 100-200ul EluentB or deionized water to each well, let stand at room temperature for 2 minutes, and seal the 96 with another new BF-400 membrane. Well DNA plate, centrifuge at 6000 rpm for 4 min to elute the DNA.
