Blood Genomic DNA Midi Kit（1-5 ml）

Item Number

B669892

• Storage Temp: Store at 2-8°C
• Shipped In: Wet ice

Product Description

Products 

B669892	Component	50 T	Storage
B669892A	Buffer RCL	3×260 mL	2-8℃
B669892B	Buffer GR	25 mL	RT
B669892C	Buffer GL	25 mL	RT
B669892D	Buffer GW1 (concentrate)	13 mL	RT
B669892E	Buffer GW2 (concentrate)	15 mL	RT
B669892F	Buffer GE	15 mL	RT
B669892G	Proteinase K	50 mg	RT
B669892H	Proteinase K Storage Buffer	5 mL	RT
B669892I	Spin Columns DL with Collection Tubes	50 sets	RT

Products

This kit is suitable for the extraction of total DNA, including genomic DNA, mitochondrial DNA and viral DNA, from fresh or frozen whole blood (blood samplestreated with anticoagulants such as citrate, EDTA or heparin), plasma, serum, haematocrit brown and yellow layers, bone marrow, cell-free body fluids, etc. Theproduct can process 1-5 ml of whole blood, and can be purified to obtain sizes rangingfrom 100bp to 50kb. The purified DNA is of high yield and good quality, with maximumremoval of proteins, pigments, lipids and other inhibitory impurities, and can bedirectly used in PCR, fluorescence quantitative PCR, enzyme digestion and SouthernBlot.

Self-contained reagent: anhydrous ethanol.

Pre-experiment Preparation and Important Notes

1. Add 5ml Proteinase K Storage Buffer to Proteinase K to dissolve it, and storeit at -20℃. Do not leave the prepared Proteinase K at room temperature for a longtime, and avoid repeated freezing and thawing to avoid affecting its activity.

2. Repeated freezing and thawing of the sample should be avoided, as this may resultin smaller DNA fragments and a decrease in the amount of extracted DNA. 3.This kit can extract up to 1-5 ml of whole blood samples, if you need to extracta large number of blood samples, please use the blood genome non-column extractionkit. 

4. Anhydrous ethanol should be added to Buffer GW1 and Buffer GW2 according to theinstructions on the label of the reagent bottle before first use.

5. Please check Buffer GL for crystallization or precipitation before use, if thereis any crystallization or precipitation, please put it in 56℃water bath to re-dissolve.

6. If the downstream experiments are sensitive to RNA contamination, 4μl of DNaseFree RNase A (100mg/ml) can be added, RNase A is not provided in the kit, and canbe ordered separately from our company if needed.

7. The Buffer RCL in the kit cannot be used further after turbidity.

procedure

1. Add 1-5 ml of blood sample to a centrifuge tube (supplied) and add 3 times thevolume of Buffer RCL and gently vortex or invert to mix.

2. Centrifuge at 3000 rpm (~900 x g) for 10 minutes and carefully aspirate thesupernatant.

3. Add 400 μl Buffer GR to the precipitate and resuspend the precipitate. Note: If the downstream assay is sensitive to RNA, add 4 μl of RNase A (100 mg/ml)solution, shake for 15 seconds, and leave at room temperature for 5 minutes.

4. For 1-2 ml blood sample extraction, add 40μl Proteinase K to the above solutionand mix well; for 2-5 ml blood sample extraction, add 100μl Proteinase K to theabove solution and mix well.

5. Add 400 μl of Buffer GL, mix upside down 15 times, and vigorously vortex andshake for at least 1 minute. Note: Do not add Proteinase K directly to Buffer GL.

6. Incubate at 70°C for 10 minutes, during which time mixing was inverted severaltimes.

Note: 1) If the solution is not completely clear, add appropriate amount of Proteinase K and incubate. Extend the incubation time until the solution is completely clear. 2) The yield of DNA has been maximized by 10 minutes of incubation, and continuedprolongation of the incubation time has no effect on DNA yield or purity.

7. Add 400 μl of anhydrous ethanol and mix upside down 10 times. Centrifuge brieflyto concentrate the liquid on the walls and cap to the bottom of the tube.

8. Add all of the solution obtained in the previous step to the Spin Columns DL inthe collection tube. If the solution cannot be added all at once, transfer it severaltimes. centrifuge at 12,000 rpm (~13,400 x g) for 1 minute, pour off the waste liquidfrom the collection tube, and put the column back into the collection tube.

9. Add 500 μl of Buffer GW1 to the adsorption column (check that anhydrous ethanolis added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquidin the collection tube, and put the adsorption column back into the collection tube.

Note: It is recommended that step 9 be repeated if the sample being extracted isthe blood genome of a species such as mice or monkeys from which hemoglobin isdifficult to remove.

10. Add 500 μl Buffer GW2 to the adsorption column (check that anhydrous ethanolis added before use), centrifuge at 12,000 rpm for 1 minute, pour off the waste liquidin the collection tube, and put the adsorption column back into the collection tube.Note: Step 10 can be repeated if further DNA purity is required.

11. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in thecollection tube. Leave the adsorption column at room temperature for several minutesto dry thoroughly. Note: The purpose of this step is to remove residual ethanol from the adsorptioncolumn, which can interfere with subsequent enzymatic reactions (digestion, PCR,etc.)

12. Place the adsorption column in a new centrifuge tube, add 50-200 μl of BufferGE or sterilized water to the middle of the adsorption column overhanging the column,leave it at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 1 minute,collect the DNA solution, and store the DNA at -20℃.

Note: 1) If the downstream experiment is sensitive to pH or EDTA, you can use sterilized water for elution. The pH of the eluent has a great influence on theelution efficiency, if water is used as the eluent should ensure that its pH is7.0-8.5 (you can use NaOH to adjust the pH of the water to this range), and the elutionefficiency is not high when the pH is lower than 7.0.

2) Incubation at room temperature for 5 minutes prior to centrifugation increasesyield.

3) Re-elution with an additional 50-200 μl Buffer GE or sterilized water can increase the yield.

4) If the final concentration of DNA is to be increased, the DNA eluate obtainedin step 12 can be re-spiked onto the adsorbent membrane and centrifuged at 12,000rpm. 1min; if the elution volume is less than 200μl, the final concentration of DNA canbe increased, but the total yield may be reduced. If the amount of DNA is less than1 μg, elution with 50 μl Buffer GE or sterilized water is recommended.

5) Because DNA preserved in water is subject to acidic hydrolysis, for long-termstorage, it is recommended that it be eluted with Buffer GE and stored at -20℃.