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Get pure RNA Kit

Item # Unit Size
GK04-05
50 samples

For Research Use Only Products


  • No phenol or chloroform required
  • Short RNA isolation time
  • No need for spin colums or Filtration tubes
  • RNA recovery from a wide range of sample volumes:up to 5x108 cells, 1.5g tissue or 1 ml whole blood for GK04-05

    Application: Total RNA isolation from cell, tissue and blood

MSDS

Contents of the Kit

Lysis buffer :13 ml x 2 bottles Precipitation solution I :13 ml x 1 bottle 
DNase :0.55 ml x 1 tube Precipitation solution II :8 ml x 1 bottle 
DNase dilution buffer :12 ml x 1 bottle   

Storage Condition: 0-5ºC
Shipping Condition: ambient temperature

Required Equipment and Materials
microcentrifuge, vortex mixer, 100 μl and 1000 μl adjustable pipettes, ethanol, ß-mercaptoethanol, homogenizer (for tissue)

Product Description
Get pureRNA Kit enables quick isolation and purification of total RNA from animal cells, tissue samples, and whole blood. This kit does not use harmful organic solvents such as phenol or chloroform. Unlike the CsCl method, this kit does not require ultracentrifugation. Get pureRNA Kit can be used for a wider range of sample volumes than silica-based spin column kits. The isolated RNA can be used for downstream experiments such as RT-PCR, Northern blotting, and cDNA synthesis


Protocol for 25 to 30 mg Tissue Sample
1. Transfer 25-30 mg of chopped tissue sample pieces into 1.5 ml tube. Add 500 μl Lysis buffer and 5 μl ß-Mercaptoethanol (not included in this kit). Cool the sample in an ice bucket.
2. Homogenize the sample in the ice bucket using a homogenizer for 30-60 seconds.
3. Add 100 μl Precipitation solution I and vortex for 5 seconds.
4. Centrifuge at 12,000-14,000 rpm for 5 minutes. Transfer the supernatant to a new 1.5 ml tube. To avoid contamination, do not disturb the white precipitate during the supernatant transfer. If white precipitate remains in the transferred supernatant, repeat this step.
5. Add an equal volume of ethanol to the supernatant and vortex for 5 seconds.
6. Centrifuge at 12,000-14,000 rpm for 2 minutes. Carefully remove the supernatant without disturbing the precipitate. A white pellet should be visible on the side or bottom of the tube. Carefully remove as much of the supernatant as possible.
7. Add 1.2 ml 70% ethanol and vortex for 5 seconds.
8. Centrifuge at 12,000-14,000 rpm for 2 minutes and discard the supernatant. A white pellet should be on the side or bottom of the tube. Carefully remove as much of the supernatant as possible.
9. Prepare 20X diluted DNase working solution in a separate tube using DNase dilution buffer. (e.g., 10 μl DNase + 190 μl DNase dilution buffer).
10. Add 200 μl DNase working solution to the tube from step 8, and dissolve the pellet.
11. Incubate at 37ºC for 15 minutes.
12. Add 50 μl Precipitation solution I and vortex for 5 seconds. White precipitate should appear immediately.
13. Add 50 μl Precipitation solution II and vortex for 5 seconds. More white precipitate should appear.
14. Centrifuge at 12,000-14,000 rpm for 5 minutes. Transfer the supernatant to a new 1.5 ml tube. To avoid contamination, do not disturb the white precipitate during the supernatant transfer. If white precipitate remains in the transferred supernatant, repeat this step.
15. Add an equal volume of ethanol to the supernatant and vortex for 5 seconds.
16. Centrifuge at 12,000-14,000 rpm for 2 minutes. Carefully remove the supernatant without disturbing the precipitate. A white pellet should be visible on the side or bottom of the tube. To avoid contamination, do not disturb the white precipitate during the removal of supernatant.
17. Add 1.2 ml 70% ethanol and vortex for 5 seconds.
18. Centrifuge at 12,000-14,000 rpm for 2 minutes and discard the supernatant. A white pellet should be visible on the side or bottom of the tube. Carefully remove as much of the supernatant as possible.
19. Dissolve the pellet in RNase-free water (DEPC-treated water) or RNase-free buffer.
Table 1 RNA Recovery

The A260 nm/A280 nm ratio of the recovered RNA is between 2.0-2.2. The A260/A280 values are based on recovered RNA dissolved in TE buffer (10 mM Tris, 1mM EDTA, pH 8.0). The use of DEPC-treated water may lower the A260/A280 values.

Troubleshooting
No or low RNA recovery
Dissolve the samples compeletely with pepietting. Make sure that the RNA pellet is on the side or bottom of the tube before discarding the supermatant.


Difficulty dissolving the tissue sample
Mince or chop the tissue sample into very small pieces before transferring it to a tube.


The precipitates are not packed tightly after adding Precipitation solution II
Completely dissolve the sample before adding Precipitation solution
I. Mix the solution thoroughly by vortexing after adding Precipitation solutions I and II. Increase the centrifugation time if  12,000-14,000rpm (10,000 g) centrifugation is difficult.


Low RNA purity
Do not disturb the white precipitate during the supernatant transfers. Use a small volume micropipette (<20 ml) when removing the supernatants after the ethanol precipitation.


Degradation of isolated RNA
Use freshly prepared samples. Make sure that all materials and equipment are RNase-free (e.g., ethanol, microtubes, pipette tips).