Description

Cat No.: 1200R

Package: 50T/100T

Kit Contents：

Reagents Required But Not Provided

Absolute ethyl alcohol, add 60ml to 15ml washing buffer before use.

Protocol

1. Homogenizing                                                                                                                                                                                                              Plant Sample: Place fresh or -70°C freezing 100mg samples in liquid nitrogen and grind thoroughly with a mortar and pestle, transfer the sample powder into 1mL lysis                                                                                                                                                        Animal Tissue: Add 1ml lysis buffer to fresh or -70°C freezing 100mg samples and grind thoroughly with a mortar and pestle or homogenize with a                                                                                                                                                                                              Adherent Cell: Add 1mL lysis buffer per 106 cells in the culture dish. Pipette the lysis buffer up and down several                          Suspension Cell: Harvest cells by centrifugation. Add 1 ml of lysis buffer per 106 cells from animal, plant or yeast, or 1 × 107 cells of Blood: Take 0.2-1mL fresh blood, and add three times volumes(0.6-3mL) of lysis buffer. Mix Incubate for 10 minutes at room temperature. Centrifuge the sample at 10000rpm for 1min and discard supernatant. If precipitation contains red cells, add 2 times volume of lysis buffer and lyse again as above steps. Add 1 ml lysis buffer to precipitated mixture after centrifugation and mix thoroughly.
2. Incubate homogenized samples at room temperature for 5 min to permit complete dissociation of the nucleoprotein complex.
3. Add 2mL of chloroform to homogenized samples. Cap the tube securely and vortex for 15s. Incubate for 3-5 minutes at room temperature.
4. Centrifuge the sample at 12,000 rpm for 10 min at 2-8°C. RNA remains mainly in the aqueous Pipette the aqueous phase out into a new tube. Be careful not to absorb the precipitation.
5. Preparation of RNase-free adsorption column: Add 500µL balance buffer in adsorption column, incubate for 2 min at room Centrifuge at 12,000 rpm for 2 min at 2-8°C. Discard the remain liquid.
6. Add 200µL ethanol in the aqueous phase from step 4 and votex, tansfer the aqueous phase in adsorption column and stay for 2 min. Centrifuge at 12,000 rpm for 2 min at 2-8°C and discard the remain
7. Add 600µL washing buffer (Ensure that ethanol is added before use) in adsorption column, centrifuge at 12,000 rpm for 2 min at 2-8°C and discard the remain
8. Add 600µL washing buffer in adsorption column, centrifuge at 12,000 rpm for 2 min at 2-8°C and discard the remain
9. Centrifuge for 2 min at 12,000 rpm and remove the collection tube, open the cap and stay for a few minutes to dry the adsorption column membrane, ensure that no ethanol is carried over during RNA

Notes

1. All related vessels and consumables should be RNase-free products, operating process carefully. Wear gloves and mask when handling RNA and all reagents, as skin is a common source of
2. OD value of RNA in the aqueous solution may be between 5and 1.9, that doesn’t mean RNA contaminated, need electrophoresis ensure.