FlexGen Blood DNA Kit（0.1-20 ml）

Item Number

F669942

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

Product Description

F669942	Component	200 mL	Storage
F669942A	Buffer FG1	2×260 mL	2-8℃
F669942B	Buffer FG2	120 mL	RT
F669942C	Buffer GE	60 mL	RT
F669942D	Proteinase K	12.5 mg	RT
F669942E	Proteinase K Storage Buffer	1.25 mL	RT

Products

This kit provides a rapid and flexible method for the extraction of total DNA,including genomic DNA and mitochondrial DNA, from fresh or frozen whole blood (samples treated with anticoagulants such as citrate, EDTA, or heparin).The productcan flexibly process 0.1-20 ml of whole blood by using a non-centrifugal columnmethod without the need for organic solvents such as phenol, chloroform, and otherorganic solvents, and effectively removes proteins, pigments, lipids, and otherinhibitory impurities contamination. , lipids and other inhibitory impurity contamination. The whole process is operated in one tube, which reduces the riskof contamination and sample mix-up. The extracted DNA is of high yield and goodquality, and can be directly used in PCR, fluorescence quantitative PCR, enzymedigestion and Southern Blot, and library construction experiments. 

Product Features 

-High purity: the extracted genomic DNA can be directly used in various experimentssuch as downstream PCR, fluorescent quantitative PCR, enzyme digestion and so on.

-Large extraction volume: DNA can be extracted from 0.1-20 ml of whole blood withoutthe use of organic solvents such as phenol and chloroform.

-Strong compatibility: Suitable for processing a wide range of blood and cell samples. 

Self-contained reagents: isopropyl alcohol, 70% ethanol. 

Pre-experiment Preparation and Important Notes

1. Add the specified amount of Proteinase K Storage Buffer to Proteinase K to dissolveit and store it at -20℃. Do not leave the prepared Proteinase K at room temperaturefor a long time, and avoid repeated freezing and thawing to avoid affecting itsactivity.

2. All centrifugation operations are done at room temperature.

3. Repeated freezing and thawing of blood samples will result in smaller DNA fragments and a decrease in the amount of DNA extracted. Repeated freezing andthawing of the resulting genomic DNA should also be avoided as much as possible toavoid degradation. If genomic DNA from frozen blood is extracted, it is recommendedto thaw it quickly in a 37°C water bath before subsequent operations.

4. Storage of blood samples: 

1) Short-term storage: Blood samples with anticoagulant added can be stored at 2-8°C for up to 10 days. For some experiments, such as Southern hybridization, wherecomplete full-length genomic DNA is required, please store the blood samples at 2-8°C for no more than 3 days, when the degradation of genomic DNA is less severe.

Long-term storage: Blood with anticoagulant should be stored at -70℃(if highmolecular weight DNA is extracted, we recommend using EDTA as anticoagulant). procedure

I. Genome extraction from 100-900µl of whole blood (take 300µl of blood processingvolume as an example)

1. Take 300µl of whole blood into a 2ml centrifuge tube (prepared by yourself),add 300µl (equal volume of the sample) of Buffer FG1, mix up and down for 5 times,centrifuge at 10,000×g for 30 seconds, discard the supernatant.

2. Add 450 μl (1.5 times the sample volume) of Buffer FG1 to the centrifuge tubeand vortex to disperse the precipitate completely. centrifuge at 10,000 g for 30seconds, discard the supernatant and place the tube upside down on a clean absorbentpaper and leave for 2 minutes. Note: In rare cases the precipitate may relax, so pour off the supernatant slowly.Inverting the centrifuge tube on blotting paper reduces the reflux of supernatantfrom the walls of the tube.

3. Prepare a mixture of Buffer FG2 and Proteinase K according to the attached table(100:1 ratio). 

Note: It is best to prepare this mixture now and use it up within 1 hour after preparation. 

4. Add 150 μl of Buffer FG2 with Proteinase K and vortex immediately until thesolution is free of clumps. 

Note: 1) If more than one sample is being processed atthe same time, vortex and shake immediately after adding the Buffer FG2/ProteinaseK mixture, do not wait until all samples have been added. 

2) Usually, vortexing 3-4 times, each time for 5 seconds, can make the precipitatefully suspended, if you find that the precipitate contains colloidal material aftervortexing, you can add 30 μl of Buffer FG2/Proteinase K mixture, and vortex again.

5. Incubate at 65°C for 10 minutes, mixing several times.

Note: If the color of the sample changes from red to olive green, the protein digestion is complete.

6. Add 150 μl of isopropanol and mix thoroughly, upside down, until filamentousor clustered genomic DNA appears.

Note: It is very important to mix thoroughly with isopropanol to precipitate theDNA. If the sample has a low leukocyte content and the DNA may not be visible, turnthe tube upside down at least 20 times to ensure complete precipitation.

7. Centrifuge at 10,000 x g for 5 minutes.

Note: If the precipitate is not firmly adherent to the wall, the centrifugationtime can be extended or the centrifugation force can be increased as appropriate.

8. Discard the supernatant and place the tube upside down on clean blotting paperto drain.

Note: In some cases, the precipitate may not stick to the wall, so be careful notto aspirate the precipitate.

9. Add 300μl of 70% ethanol, vortex for 5 seconds, centrifuge at 10,000 x g for5 minutes and discard the supernatant.

Note: If the precipitate is not firmly attached to the wall, extend the centrifugation time or increase the centrifugation force appropriately.

10. Invert the centrifuge tube onto clean blotting paper for 5 minutes to ensurethat the precipitate is in the tube.

Note: A white DNA precipitate is visible at the bottom of the tube and in rare casesthe precipitate may be slack, so pour off the supernatant slowly.

11. Air dry the DNA precipitate until all liquid evaporates (at least 5 minutes).

Note: Ethanol residue can affect subsequent enzyme reactions (digestion, PCR, etc.),but avoid over-drying the DNA precipitate as over-drying will make the DNA difficultto dissolve.

12. Add 200 μl Buffer GE, vortex at low speed for 5 seconds, and incubate at 65°C for 1 hour to dissolve the DNA, flicking several times during the period to aiddissolution. Store the DNA at -20°C.

Note: If the DNA is not completely solubilized, it can be left at room temperatureovernight. 

II.Extraction of genome from 1-5 ml of whole blood (take 3 ml of blood processingvolume as an example) 

1. Take 3 ml of whole blood in a 15 ml centrifuge tube (self-provided), add 3 ml(equal volume of the sample) of Buffer FG1, mix up and down for 5 times, centrifugeat 2,500×g for 5 minutes, and discard the supernatant. 

2. Add 4.5 ml (1.5 times the sample volume) of Buffer FG1 to the centrifuge tube,vortex and shake to disperse the precipitate completely. centrifuge for 5 minutesat 2,500 × g, discard the supernatant, and place the tube upside down on cleanabsorbent paper and leave it for 2 minutes. Note: In rare cases the precipitate may relax, so pour off the supernatant slowly.Inverting the tube onto blotting paper will minimize reflux of supernatant from thetube wall.

3. Prepare a mixture of Buffer FG2 and Proteinase K (100:1) according to the attachedtable. 

Note: It is best to use this mixture as it is, and to use it up within 1 h after preparation.

4. Add 1.5 ml of Buffer FG2/Proteinase K mixture and vortex immediately until thesolution is free of clumps. 

Note: 1) If more than one sample is being processed at the same time, vortex andshake immediately after adding Buffer FG2/Proteinase K mixture, do not wait untilall samples have been added.

2) Usually, vortexing 3-4 times, each time for 5 seconds, can make the precipitatefully suspended, if you find that the precipitate contains colloidal material aftervortexing, you can add 300 μl of BufferFG2/Proteinase K mixture, and then vortexagain to mix well. 

5. Incubate at 65°C for 10-30 minutes, mixing several times.

Note: If the color of the sample changes from red to olive green it means the proteindigestion is complete.

6. Add 1.5 ml of isopropanol and mix thoroughly by turning up and down until DNAis visible.

Note: It is important to mix thoroughly with isopropanol to precipitate the DNA.If the sample is low in leukocytes and the DNA may not be visible, turn the tubeupside down at least 20 times to ensure complete precipitation.

7. Centrifuge at 2,500 x g for 5 minutes.

Note: If the precipitate is not firmly adherent to the wall, the centrifugationtime can be extended or the centrifugation force increased as appropriate.

8. Discard the supernatant and place the tube upside down on clean blotting paperto drain.

Note: White DNA precipitate can be seen at the bottom of the tube. In rare cases,the precipitate may be loose, so pour off the supernatant slowly.

9. Add 1.5 ml of 70% ethanol, vortex and shake for 5 seconds, centrifuge at 2,500x g for 5 minutes and discard the supernatant.

Note: If the precipitate is not firmly attached to the wall, you can extend thecentrifugation time or increase the centrifugal force appropriately.

10. Invert the centrifuge tube onto clean blotting paper for 5 minutes to ensurethat the precipitate is in the tube.

Note: In rare cases the precipitate may be slack, so pour off the supernatant slowly.

11. Air dry the DNA precipitate until all liquid has evaporated (at least 5 minutes).

Note: Ethanol residues can affect subsequent enzymatic reactions (digestion, PCR,etc.), but avoid over-drying the DNA precipitate, as over-drying can make the DNAdifficult to dissolve.

12. Add 300μl of Buffer GE, vortex at low speed for 5 seconds, incubate at 65℃for 1 hour to dissolve the DNA, and flick several times during the incubation toaid in dissolution. Store the DNA at -20°C. 

Note: If the DNA is not completely solubilized, it can be left at room temperatureovernight.

III.Extraction of genome from 6-20 ml of whole blood (take 10 ml of blood processingvolume as an example) 

1. Take 10 ml of whole blood in a 50 ml centrifuge tube (self-provided), add 10ml of Buffer FG1, mix it up and down for 5 times, and centrifuge it at 2,500×g for5 minutes.

2. Add 15 ml of Buffer FG1 to the centrifuge tube, vortex and shake to dispersethe precipitate completely. centrifuge for 5 minutes at 2,500 x g. Discard thesupernatant and place the tube upside down on clean blotting paper and leave for2 minutes. 

Note: In rare cases the precipitate may relax, so pour off the supernatant slowly.

3. Prepare a mixture of Buffer FG2 and Proteinase K according to the attached table(100:1 ratio).

Note: It is best to prepare this mixture now and use it up within 1h after preparation.

4. Add 5 ml of Buffer FG2/Proteinase K mixture and vortex immediately until thesolution is free of clumps.

Note: 1) If more than one sample is handled at the same time, vortex and shakeimmediately after adding Buffer FG2/Proteinase K mixture, do not wait until allsamples have been added.

2) Usually, vortexing and shaking 3-4 times for 5 seconds each time can make theprecipitate fully suspended. If the precipitate contains gelatinous material aftervortexing and shaking, add 1ml of BufferFG2/Proteinase K mixture and vortex againto mix well. 

5. Incubate at 65°C for 30 minutes, mixing several times.

Note: If the color of the sample changes from red to olive green, the proteindigestion is complete.

6. Add 5 ml of isopropanol and mix thoroughly by turning up and down until DNA isvisible. 

Note: It is very important to mix thoroughly with isopropanol to precipitate theDNA, and the tube should be turned upside down at least 20 times to ensure completeprecipitation.

7.2 Centrifuge at 500 x g for 5 minutes. 

Note: If the precipitate is not firmly adherent to the wall, the centrifugationtime can be extended or the centrifugation force can be increased.

8. Discard the supernatant and place the tube upside down on clean blotting paperto drain. Note: White DNA precipitate can be seen at the bottom of the tube. In rare cases,the precipitate may be loose, so pour off the supernatant slowly.

9. Add 5 ml of 70% ethanol, vortex and shake for 5 seconds, centrifuge at 2,500x g for 5 minutes and discard the supernatant. Note that if the precipitate is not firmly adhered to the wall, you can extend thecentrifugation time or increase the centrifugal force appropriately.

10. Place the centrifuge tube upside down on clean absorbent paper for 5 minutesto ensure that the precipitate is in the tube. Note: In rare cases the precipitate may be slack, so pour off the supernatant slowly.

11. Air dry the DNA precipitate until all liquid has evaporated (at least 5 minutes).Note: Ethanol residues can affect subsequent enzyme reactions (digestion, PCR, 

etc.), but avoid over-drying the DNA precipitate as over-drying will make the DNAdifficult to dissolve.

12. Add 1 ml of Buffer GE, vortex at low speed for 5 seconds, and incubate at 65°C for 1 hour to dissolve the DNA, flicking several times during the period to aiddissolution. Store the DNA at -20°C. 

Note: If the DNA is not completely dissolved, it can be left at room temperatureovernight. 

Table: Amounts of various buffers required for different volumes of blood 

	100	300	1000	Volume of blood sample（μl） 3000 5000	10000	20000
Buffer FG1（μl） Buffer FG2（μl） Proteinase K（μl） isopropanol（μl）  70%ethanol（μl） Buffer GE（μl） Add a mixture of FG2 and Protein K	250 50 0.5 50 50 100 10	750 150 1.5 150 150 200 30	2500 500 5 500 500 200 100	7500 1500 15 1500 1500 300 300	12500 2500 25 2500 2500 500 500	25000 5000 50 5000 5000 1000 1000	50000 10000 100 10000 10000 1000 1000