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Total RNA isolation kit
Cat#GR1012   50 preps
Introduction
The kit procedure represents a well-established technology for RNA purification. This technology combines the selective binding properties of a silica membrane with the speed of microspin technology. A specialized high-salt buffer system allows up to 100g of RNA longer than 200bases to bind to the silica membrane. Biological samples are first lysed and homogenized in the presence of a highly denaturing guanidine-thiocyanateCcontaining buffer, which immediately inactivates RNases to ensure intact RNA. Ethanol is added to provide appropriate binding conditions, and the sample is then applied to an RNA spin-column, where the total RNA binds to the membrane and contaminants are efficiently removed. High-quality RNA is then eluted in 30C100 l water.
Kit Content

Component
Storage
50 preps
Buffer RL
4  
50 ml
Buffer RE
RT
25 ml
Buffer RW
RT
10 ml
Add 40ml ethanol before use.
RNase-free H2O
RT
10 ml
70% ethanol  
RT
9 ml RNase-free H2O
Add 21ml ethanol before use.
Spin-column AC(RNase-free)
RT
50
Collection Tube2ml
RT
50
Manual
 
1

All reagents, when stored properly, are stable for 12 months.
Storage Notes:
1) All reagents should be clear. In case, some may precipitate due to low temperature, please incubate them at 37 for a moment until clear, and then cool down to RT before use.
2) Some reagents will precipitate because of been stored in 4 or - 20, which will affect the yield, so incubate till no precipitation before use. All reagents can be transported under room temperature (15-25). Buffer RL can be transported under RT and keep at 4 upon arrival.
3) Please ensure the bottles tightly capped when not in use, prevent reagents from evaporating, oxidation and pH change.
Features
1) Stability, comparable RNA yield with high quality absorbing membrane.
2) High-purity, specifically membrane absorption and washing for removing protein
and other debris.
3) Unique Buffer RL can effectively eliminate genomic contamination.
Before staring
Please read this section before your experiment.
1. Please add ration ethanol to Buffer RW and 70% ethanol before first use. Mix well and mark the check box labeled on the bottles to indicate that the ethanol has been added.
2. To prevent RNA degradation, all the centrifuge steps should be made under 4, except having special notes, suggest using up to 13,000 rpm traditional centrifuge, for example Eppendorf 5415C.
3. Buffer RL and Buffer RE contain stimulating compound, please wear latex gloves, avoiding skin, eyes and cloth to be contaminated. If that, please use water or physiological saline to wash the exposed body parts.
4. Due to the prevalence of RNases, wear gloves at all times and change them whenever may have been contacted by reagents, please follow standard laboratory procedures of Molecular Clone rules.
* Wear gloves in entire process. Skin often contains bacteria and molds that can contaminate an RNA preparation and be a source of RNases.
* Use sterile, disposable plasticware and automatic pipettes reserved for RNA work to prevent cross-contamination with RNases from shared equipment. For example, a laboratory that is using RNA probes will likely be using RNase A or T1 to reduce background on filters, and any nondisposable items (such as automatic pipettes) can be rich sources of RNases.
* Treat non-disposable glassware and plastic-ware before use to ensure that it is RNase-free. Bake glassware at 200C overnight, and thoroughly rinse plastic-ware with 0.1N NaOH, 1mM EDTA followed by RNase-free water.
5. The integrity of purified RNA may be determined by denaturing agarose gel electrophoresis (or agarose gel electrophoresis). The ratio of 28S to 18S ribosomal RNA should be approximately 2:1 by ethidium bromide staining. Sometimes there may be the third band about 0.1-0.3kb (5S RNA and tRNA), even 4 or 5 bands will appear in some plant tissues. Once the preRNA, hnRNA, small RNA are extracted from the sample, you will see some discontiguous bands of 7kb-15kb. All of them are normal.
6. The most common method to determine the yield and purity of RNA is spectrophotometry (OD260/OD280). Please dissolute RNA by TE, water will make OD280 higher because of lower ion intensity and pH.
7. Prepare chloroform before use.
Protocol
Note: Add absolute ethanol to Buffer WB and 70% ethanol.
1. Homogenization:
a. Tissues
Please homogenize tissue in an appropriate volume of Buffer RL (50-100mg/mL) until no visible tissue. Pay attention to the volume of sample should not beyond 1/10 total volume of Buffer RL.
b. Cells Grown in Monolayer
You can directly append an appropriate volume Buffer RL to the culture plate for dissolve cell, and transfer dissolution by pipetting. The volume of Buffer RL is decided by the area of culture plate, about 10cm2 per 1ml. Once appending not enough Buffer RL, its possible to contaminated genomic DNA.
c. Cells Grown in Suspension
Pellet cells by centrifugation. Lyse cells in Buffer RL by repetitive pipetting. Use 1 ml of the reagent per 5-10 106 of animal, plant or yeast cells, or per 1 107 bacterial cells. Washing cells before addition of RL should be avoided as this increases the possibility of mRNA degradation. Disruption of some yeast and bacterial cells may require the use of a homogenizer.
2. Incubate the homogenized samples for 5 minutes at 15 -30C to permit the complete dissociation of nucleoprotein complexes.
3. Optional: Centrifuge at 12,000 rpm for 10 min at 4. Pipette the supernatant to a RNase-free centrifuge tube.
An additional isolation step may be required for samples with high content of proteins, fat, polysaccharides or extracellular material such as muscles, fat tissue, and tuberous parts of plants.
4. Add 0.2 ml of chloroform per 1 ml of Buffer RL. Cap sample tubes securely. Shake tubes vigorously by hand for 15 seconds and incubate them at RT for 3 minutes.
5. Centrifuge at 12,000rpm for 10 minutes at 4; (Centrifuge the samples at no more than 12,000 rpm for 15 minutes at 2~8).
6. Transfer the aqueous phase to a fresh tube. Add 1 volume 70% ethanol. Mix well (precipitate may form). Transfer the mixture and precipitate to a Spin-column AC (placed in collection tube). If the mixture is too much, apply the mixture in successive application to the same Spin-column AC.
7. Centrifuge at 10,000 rpm for 45s at 4. Discard the flow through. Reuse the Spin-column and the collection tube.
8. Add 500l Buffer RE to the center of Spin-column AC to remove the protein. Centrifuge at 12,000 rpm for 45s. Discard the flow through.
9. Add 500l Buffer RW. Centrifuge at 12,000rpm for 45s. Discard the flow through.
10. Add 500l Buffer RW. Centrifuge at 12,000rpm for 45s. Discard the flow through.
11. Replace Spin-column AC to the collection tube and centrifuge for 2min to remove the residual fluid.
12. Place Spin-column AC to a 1.5ml RNase-free centrifuge tube. Apply 50-80l RNase-free H2O (Pre-heated to 65-75 is better ) to the center of the column RA. Place it at room temperature for 2min. Centrifuge at 12,000rpm for 1min. If desired, wash the Spin-column AC with 30l RNase-free water, combining the second eluate with the first in the same Collection Tube; approximately 90% of the RNA is recovered during the first elution step.
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