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Mag-Bind Blood RNA Isolation Protocol (for 50ul Blood)

互联网2013-11-13

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实验原理

If using the Mag-Bind™ Blood RNA Kit for the first time, please read this booklet to become familiar with the procedure and its various modifications. Samples are lysed in a specially formulated buffer containing detergent and chaotropic salt. After adjust the buffer condition, nucleic acids (DNA/RNA) will form a complex with magnetic beads. The beads/nucleic acids complex is separated from lysates using a magnet. Proteins and cellular debris are efficiently washed away by a washing step. Next, DNA is removed by DNase I treatment. RNA is re-bound and cleaned from the DNase I reaction mixture using a second magnetic beads binding and washing procedure. Finally, pure RNA is then eluted in nuclease-free water or low ionic strength buffer. Purified RNA can be directly used in downstream applications without the need for further purification.

实验试剂

Regents supplied by User

1. Absolute ethanol (96-100%)

实验设备

Equipments supplied by User

1. Magnetic separation device for 96 well microplate (MSD-01)

2. Nuclease-free 96-well

3. Multichannel pipette

4. Nuclease-Free pipetting tips

实验步骤

The following protocol is designed for isolating total RNA from Fresh Whole Blood. For best RNA quality, always use blood that has not been frozen. Frozen blood can be used with this protocol, however, RNA quality could be compromised as the results of freezethawing process.

1. Prepare a Lysis Mixture by mixing one volume of Buffer MRB and one volume of isopropanol. For 96 samples, combine15 ml Buffer MRB with 15 ml isopropanol and mix throughly by vortexing. Add 300 ul Lysis Mixture for each sample at step 3.

2. Add 50 ul blood sample into Processing Plate (supplied) followed by adding 10 ul Proteinase K solution and 10 ul Mag-Bind™ Particle R.

Note: Proteinase K and Mag-Bind™ Particles R can be prepared as master mix and add to each at one time. For 96 samples, mix 1 vial Mag-Bind™ Particle R (1 ml) with 1 ml Proteinase K solution. Add 20 ul beads/proteinase K mix for each blood sample.

3. Add 300ul Lysis Mixture, shaking for 10 minutes at room temperature.

4. Place the Processing Plate on a magnetic separation device to magnetize the magnetic particles. Leave the plate on the magnet until all the magnetic particles are pelleted. The capture time is depend on the sample type and magnetic stand used. If Omega Bio-tek MSD-01 is used, it normally take 7-15 minutes.

5. Carefully aspirate and discard the supernatant without disturbing the magnetic particles.

6. Remove the Processing Plate containing the magnetic particles from the magnetic separation device. Add 300 ul of Buffer MBW and resuspend magnetic particles pellet by pipetting up and down or vortex the plate at maximum speed for 1 minute.

7. Place the plate onto a magnetic separation device to magnetize the magnetic particles. Aspirate and disacrd the cleared supernatant after the lysate is cleared.

8. Remove the plate containing the magnetic particles from the magnetic separation device. Add 400 ul of SPR Wash Buffer to each well and resuspend magnetic particles pellet by pipetting up and down or vortexing for 30 seconds.

9. Place the plate onto a magnetic separation device to magnetize the magnetic particles. Aspirate an discard the cleared supernatant after the lysate is cleared.

10. Leave the tube on the magnetic separation device. Prepare the DNase I digestion mix as following table.

Note: If total nucleic acid (DNA and RNA) is desired, skip this DNase I digestion steps (step 14-21) and proceed step 17 for isolating both DNA and RNA.

Note: If total nucleic acid (DNA and RNA) is desired, skip this DNase I digestion steps (step 14-21) and proceed step 17 for isolating both DNA and RNA.

11. Add 50 μl of DNase I digestion mix and resuspend the magnetic particles by pipetting up and down for 20 times or vortexing. Incubate at room temperature for 10-15 minutes.

Note: It is very important to remove any liquid drop from the wells of the process plate before adding the DNase I digestion mix. DNase I digestion Mix must be used immediately once it is prepared.

12. Add 300 μl Buffer MBW and mix throughly by vortexing for 3 minutes. Incubate at room temperature for 5-10 minutes.

13. Place the plate onto a magnetic separation device to magnetize the magnetic particles. Aspirate the cleared supernatant after the lysate is cleared.

14. Add 400 ul of SPR Wash Buffer and resuspend magnetic particles pellet by pipetting up and down for 20 times or vortexing at maximum speed for 30 seconds.

15. Place the plate onto a magnetic separation device to magnetize the Mag-Bind™ particles.

16. Aspirate and discard the cleared supernatant. Carefully remove any liquid drop from tube. Leave the tube on the magnet stand. Air dry the magnetic particles at room temperature for 7-10 minutes.

17. Add 20-50 μl DEPC Treated water and resuspend magnetic particles pellet by vortexing or pipetting up and down for 20 times. Incubate at room temperature for 3 minutes.

18. Place the tube onto a magnetic separation device to magnetize the Mag-Bind. particles.

19. Transfer the cleared supernatant contains purified RNA into a new RNase-free microplate .

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相关问答

问

抗体包被的protocol

问

所在实验室pcr体系常用的有20ul，50ul，能不能100ul呢？pcr管是200ul的容积

5 回答 834 围观

问

DNA提取洗脱体积太小，50ul会有什么影响？

4 回答 338 围观

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