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1. Binding solution CB or inhibitor removal solution IR may precipitate and precipitate at low temperatures. It can be re-dissolved in a water bath at 37 ° C for a few minutes. It is clear and transparent and then cooled to room temperature.
2. In order to avoid reducing the activity and convenient transportation, the proteinase K is provided as a lyophilized powder. After receiving it, it can be centrifuged briefly, and then dissolved in 1 ml of sterilized water. Because repeated freezing and thawing may reduce the enzyme activity, immediately after dissolving, it will be stored frozen at -20 ° C according to the amount of each use (20 μl).
3. Avoid prolonged exposure to air, such as volatilization, oxidation, and pH change. Cover each lid after use.
ï¶ Operation steps: (Please read the precautions before the experiment)
Tip: Before the first use, please add the specified amount of absolute ethanol in the rinse liquid WB, mix well, please add the ethanol in the box after the check, so as not to join multiple times!
1. Take 200 ul of fresh, frozen or added anticoagulant blood and place in a 1.5 ml centrifuge tube.
If the starting amount of whole blood is less than 200 μl, the buffer BB is used to make up to 200 μl. If the starting amount is between 200 μl and 300 μl, subsequent operations require a proportional increase in reagent usage. If the starting amount is between 300 μl and 1 ml, the red blood cell lysis operation needs to be performed first (see appendix after this manual).
2. Add 20 μl of proteinase K (20 mg/ml) solution, mix well, add 200 μl of binding solution CB, mix well by vortexing, and let stand at 70 ° C for 10 minutes. The solution strain is clear (but the color is black).
Optional steps, generally do not need: If there are more RNA residues, need to remove RNA, you can add 20μl RNase A (25mg / ml) solution before adding 200μl binding solution CB, shake and mix, let stand for 5-10 minutes at room temperature.
3. After cooling, add 100 μl of isopropanol and mix immediately by vortexing. At this point, flocculation may occur.
It is very important to vortex or blow directly and mix well in the above steps. The mixing is not enough to seriously reduce the yield. If necessary, if the sample is sticky and difficult to mix, it can be vortexed for 15 seconds to mix.
4. Add the mixture from the previous step (including possible precipitation) to an adsorption column AC (in the collection tube) and centrifuge at 13,000 rpm for 30-60 seconds, and drain the waste from the collection tube.
5. Add 500 μl of inhibitor-removal IR, centrifuge at 12,000 rpm for 30 seconds, and discard the waste.
6. Add 700 μl of rinse WB (check to see if absolute ethanol has been added!), centrifuge at 12,000 rpm for 30 seconds, and discard the waste.
7. Add 500 μl of rinse WB, centrifuge at 12,000 rpm for 30 seconds, and discard the waste.
8. Place the adsorption column AC back into the empty collection tube and centrifuge at 13,000 rpm for 2 minutes to remove the rinse solution as much as possible to prevent residual ethanol in the rinse solution from inhibiting the downstream reaction.
9. Remove the adsorption column AC and place it in a clean centrifuge tube. Add 100 μl of elution buffer EB to the middle of the adsorption membrane. (The elution buffer is better preheated in a 65-70 ° C water bath beforehand.) Leave for 3-5 minutes and centrifuge at 12,000 rpm for 1 minute. The resulting solution was re-introduced into a centrifugal adsorption column, allowed to stand at room temperature for 2 minutes, and centrifuged at 12,000 rpm for 1 minute.
The larger the elution volume, the higher the elution efficiency. If the DNA concentration is high, the elution volume can be appropriately reduced, but the minimum volume should not be less than 50 μl. The volume is too small to reduce the DNA elution efficiency and reduce the DNA yield.
10. DNA can be stored at 2-8 ° C, if you want to store for a long time, can be placed at -20 ° C.
附录 Appendix (for example, red blood cell lysis operation with 300 μl, 1 ml whole blood):
1. Pipette 900 μl of red blood cell lysate into a 1.5 ml centrifuge tube or 3 ml red blood cell lysate into a 15 ml centrifuge tube. (Red blood cell lysate can be purchased from our company)
2. Invert the anticoagulated whole blood (return to room temperature before use), then add 300μl whole blood and 1ml whole blood to the above 1.5ml and 15ml centrifuge tubes, invert 6-8 times, and invert the flick tube. Wall, make sure to mix well.
3. Leave at room temperature for 10 minutes (during a period of time by mixing the flicks several times to help lyse the red blood cells).
4. Centrifuge at 12,000 rpm for 20 seconds (for 1.5 ml centrifuge tubes) or 2,000-3,000 rpm for 5 minutes (for 15 ml centrifuge tubes), discard the red supernatant, and carefully discard as much of the supernatant as possible (be careful not to suck To the cell mass at the bottom of the tube, leave a complete tube bottom white blood cell mass and approximately 10 μl of residual supernatant.
After centrifugation, white leukocyte clusters should be seen at the bottom of the tube. There may also be some red blood cell debris and white blood cell clusters together. However, if most of the red cell clusters are seen, the red blood cell lysis is not sufficient, and red blood cell lysis should be added. Repeat steps 3 and 4 after resuspending the cell pellet.
5. Add 200 μl of buffer BB to vortex and resuspend the leukocyte mass to fully disperse the leukocyte mass.
Among them, hepatocyte anti-coagulation white blood cell sedimentation group is difficult to disperse and resuspend, affecting the subsequent experimental lysis effect, it is recommended to use non-heparin anticoagulant to collect blood samples.
6. The whole blood genomic DNA can now be extracted according to the procedure.