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E.Z.N.A.® Mag-Bind® Viral DNA/RNA Spin Protocol

互联网2013-11-13

758

实验原理

If using the E.Z.N.A.™ Mag-Bind® Viral DNA/RNA Kit for the first time, please read this booklet to become familiar with the procedure and its various modification. Samples are lysed in a specially formulated buffer containing detergent. DNA/RNA was bound to the surface of Mag-Bind® magnetic particles under proper condition. Proteins and cellular debris are efficiently washed with few wash steps. Pure RNA and DNA is then eluted in nuclease-free water or low ionic strength buffer. Purified RNA or DNA can be directly used in downstream applications without the need for further purification.

实验试剂

1. Mag-Bind® Magnetic Beads R 2.1 ml 8.4 ml

2. QVL2 Lysis Buffer 50 ml 200 ml

3. Glycogen 0.8 ml 2 x 1.5 ml

4. Buffer VHB 34 ml 2 x 66 ml

5. SPR Wash Buffer 4 x 12 ml 4 x 20 ml

6. Nuclease free Water 10 ml 45 ml

实验设备

Purification 1 x 96 Preps 4 x 96 Preps

实验步骤

1. Distribute 150 ul sample into a 1.5 ml tube or a 96 deep well plate. If using pre-frozen samples, thaw at room temperature (15-20℃) and mix well by shaking or pipetting before proceeding step 2.

2. Add 300 ul of QVL Lysis Buffer, 6ul Glycogen and 20ul Mag-Bind® Magnetic Beads R to the sample. Vortex at maxi speed for 30 seconds. Incubate at room temperature for 10 min.

Note: Add 20 ul 2-mercaptoethanol per 1 ml of QVL Lysis Buffer before use. This mixture can be made and stored at room temperature for 1 week.

3. Add 500ul Isopropanol followed with mix thoroughly by vortexing or pipetting up and down 30 times. Incubate at room temperature for 10 minutes.

4. For 1.5 ml tube, centrifuge at 10,000 x g for 5 min, or centrifuge at 4000 x g for 10 min for 96-well plate. Remove and discard the supernatant.

5. Add 0.5ml Buffer VHB to each sample. Vortex to completely resuspend magnetic paritcles.

Note: Buffer VHB must be diluted with absolute ethanol before use. Refer to label on bottle for instruction.

6. Centrifuge at 10,000 x g for 5 min for 1.5ml tube, or centrifuge at 4000 x g for 10 min for 96-well plate. Remove and discard the supernatant.

7. Add 0.7ml SPR Wash Buffer to each sample. Vortex to completely resuspend magnetic paritcles.

8. Centrifuge at 10,000 x g for 5 min for 1.5ml tube, or centrifuge at 4000 x g for 10 min for 96-well plate. Remove and discard the supernatant.

9. Repeat step 7-8 by adding another 0.7 ml SPR Wash Buffer to each sample.

10. Remove and discard the supernatant. Briefly spin to collect the liquid into the tube bottom. Remove any of liquid by pipettor and air dry 10-15 min.

11. Add 20-50ul nuclease free water, vortex to suspend magnetic beads completely. Incubate 3-5 minutes at room temperature. Centrifuge at 10000xg for 2 min, or centrifuge at 4,000 x g for 10 min. Transfer solution into a new tube or a new 300ul 96-well plate.

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