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Plant Total RNA Isolation Kit Plus is suitable for the isolation of total RNA from 50-200 mg plants.Plant tissue is extracted with lysate and Buffer EX to obtain RNA-containing supernatant, which is added to a spin column after adding ethanol. RNA is bound to the spin column, and dissolved proteins and PCR inhibitors are filtered out. RNA is washed by two washing solutions and eluted with RNase-free Water, and then it can be used in various molecular biology experiments such as RT-PCR, Northern blot, Dot blot, mRNA isolation, etc.Product Composition
Plant Total RNA Isolation Kit Plus Cat. No. | 5preparations 5103005 | 50preparations 5103050 |
Filter Columns Spin Columns β-mercaptoethanol Buffer RCT Buffer EX Buffer K Buffer WA(concentrate) Buffer WBR(concentrate) RNase-free Water Instructions | 5 sets 5 sets 50 μl 4 ml 4 ml 2 ml 1.9 ml 1.5 ml 1.5 ml 1 copy | 50 sets 50 sets 500 μl 32 ml 32 ml 20 ml 12 ml 10 ml 2 ml×3 1 copy |
Product Storage and Expiry Date
If the kit is stored at room temperature (0-30℃), the performance of the kit can be maintained without significant change within two years; if the product is stored at 2-8℃, the validity period of the product can be extended to more than two years.
Product Description
This product is suitable for the isolation of total RNA from 50-200 mg plants. Plant tissue is extracted with lysate and Buffer EX to obtain RNA-containing supernatant, which is added to a spin column after adding ethanol. RNA is bound to the spin column, and dissolved proteins and PCR inhibitors are filtered out. RNA is washed by two washing solutions and eluted with RNase-free Water, and then it can be used in various molecular biology experiments such as RT-PCR, Northern blot, Dot blot, mRNA isolation, etc.
Equipment and Reagents to Be Supplied by User
1． Absolute Ethanol and 70% Ethanol
2． RNase-free 1.5 ml microcentrifuge tubes
3． Pipettes and tips (in order to avoid RNase contamination, it is recommended to use RNase-free pipette tips with filters)
4． Protective equipment such as disposable latex gloves and paper towels
5． Microcentrifuge(s) (with rotor for 1.5 ml and 2 ml microcentrifuge tubes)
6． Vortexer
7． Laboratories that do not use RNase
Preparation Before Use
1. Check whether there is precipitate in Buffer RCT. If there is precipitate, please incubate at 60℃ to dissolve it before use.
2. If the centrifuge has refrigeration function, please set the temperature to 25℃.
3. Add 10 µl β-mercaptoethanol to every 1 ml Buffer RCT and mix well. The use of Buffer RCT with β-mercaptoethanol within one month does not affect the experimental results.
4. Add absolute ethanol to Buffer WA and Buffer WBR according to the instructions on the label of the reagent bottle, and tick the box on the label to mark "Alcohol added".
5. Because saliva and skin contain RNase, latex gloves and masks are required during the whole process of RNA extraction.
6. If DNA needs to be completely removed in subsequent experiments, please add a DNase I digestion step, and the DNase I On-column Digestion Kit (Cat. No. 8010050) needs to be purchased separately.
Protocol:
1. Weigh 200–500 mg of plant tissue in a mortar, add liquid nitrogen, grind the tissue into a powder, and weigh 50–200 mg of the tissue ground into a powder into a 1.5 ml microcentrifuge tube pre-cooled with liquid nitrogen .
* When grinding tissue, liquid nitrogen should be added in time to avoid tissue melting, so as to prevent endogenous RNase from reactivating and degrading RNA.
* Only samples with low RNA content (such as potato tubers, watermelon pulp, etc.) are recommended to use 200 mg of tissue; for samples with high RNA content such as young leaves and shoots, the dosage should be controlled within 100 mg, otherwise it may clog the filter column or spin column; the nucleic acid content in fresh shoots is high, and there may be a lot of genomic DNA remaining, so DNase I digestion is recommended.
2. Add 600 µl Buffer RCT which β-mercaptoethanol has been added, and vortex until the tissue is completely lysed .
3. Add 600 µl Buffer EX, mix vigorously, and centrifuge at 12000 rpm for 5 minutes.
4. Carefully pipette 350 µl supernatant and transfer to a clean RNase-free 1.5 ml microcentrifuge tube.
* Be careful not to bring in the protein precipitate in the middle.
5. Add 350 µl Buffer K to the supernatant and mix well by pipetting 6 - 8 times. Transfer the mixture to a filter column, close the lid and centrifuge at 13000 rpm for 1 minute.
* Do not omit this step, otherwise it may cause clogging of the spin column in the subsequent operation steps.
6. Keep the collection tube and discard the filter column, add 700 µl 70% ethanol to the filtrate and mix well by pipetting 6 - 8 times, pipette 700 µl mixture into the spin column, close the lid, and centrifuge at 13000 rpm for 1 minute.
* If there is precipitation after adding 70% ethanol and mixing, please add the precipitation to the spin column together.
7. Discard the filtrate in the 2 ml collection tube, put the spin column back into the 2 ml collection tube, pipette the remaining mixture into the spin column, close the lid and centrifuge at 13000 rpm for 1 minute.
* The filtrate does not need to be completely discarded. If you want to avoid the contamination of the centrifuge by the filtrate adhering to the nozzle of the collection tube, you can slap the 2 ml collection tube upside down on a paper towel once.
8. Discard the filtrate in the 2 ml collection tube, put the spin column back into the 2 ml collection tube, add 500 µl Buffer WA to the spin column, close the lid and centrifuge at 13000 rpm for 1 minute.
* Make sure absolute ethanol has been added to Buffer WA.
9. Discard the filtrate in the 2 ml collection tube, put the spin column back into the 2 ml collection tube, add 600 µl Buffer WBR to the spin column, close the lid and centrifuge at 13000 rpm for 1 minute.
* Make sure absolute ethanol has been added to Buffer WBR.
10. Discard the filtrate in the 2 ml collection tube, put the spin column back into the 2 ml collection tube, and centrifuge at 14000 rpm for 1 minute.
* If the top speed could not reach 14000 rpm, centrifuge at full speed for 2 minutes.
* Do not omit this step, otherwise the subsequent RT-PCR effect may be affected due to the residual ethanol in the eluate.
11. Discard the 2 ml collection tube, put the spin column into a clean RNase-free 1.5 ml microcentrifuge tube, add 50-100 µl RNase-free Water to the center of the membrane of the spin column, close the lid and incubate for 1 minute at room temperature, centrifuge at 13000 rpm for 1 minute.
* If the centrifuge does not have a leak-proof cover, please change the centrifugation condition to 8000 rpm for 1 minute to prevent the 1.5 ml microcentrifuge tube cover from falling off and damaging the centrifuge.
12. Discard the spin column, and the eluted RNA can be used immediately for various molecular biology experiments; or store the RNA below -70℃ for later use.
