实验概要
This protocol is designed to clean up and concentrate RNA from various sources such as RNA isolated with RNA-solv® Reagent and other phenol involved reagents.
实验步骤
1. Measure the volume of sample and transfer into a new 1.5 ml microcentrifuge tube. Adjust the sample volume to 100ul with DEPC-Water and proceed to step 2.
Note: if the starting samples is RNA pellet, dissolve the samples with DEPC treated water.
2. Add 350ul QVL Lysis buffer and mix by vortexing. When process small amount of RNA (2 ug). Add 2 ul of Linear Acrylamide to the mixture.
Note: Remember to add 20 ul of 2-mercaptoethanol per 1 ml of QVL Lysis Buffer before use.
3. Add 250 ul absolute ethanol (96-100%, room temperature) to the sample and mix thoroughly by vortexing.
4. Apply sample from step 3 to HiBind® MicroElute RNA column inserted in a 2 ml collection tube (supplied). The maximum capacity of the spin cartridge is 700 ul. (Larger volumes can be loaded successively.) A precipitate may formupon addition of ethanol in Step 3. Vortex and add the entire mixture to the column. With the spin column inside a 2ml collection tube (supplied with kit), centrifuge at 10,000 x g for 30 seconds at room temperature. Discard flowthrough and re-use the collection tube.
5. Place column in the same 2 ml collection tube from step 4 and add 500 ul RWB Wash Buffer diluted with absolute ethanol. Centrifuge and discard flowthrough.
Note: RWB Wash Buffer Concentrate must be diluted with absolute ethanol before use. Refer to label on bottle for directions.
6. Place column back into the same collection tube from step 5, and add another 500 ul RWB Wash Buffer diluted with absolute ethanol. Centrifuge and discard the flow-through and collection tube.
7. Place the column into a new 2 ml collection tube (supplied). Centrifuge the column at full speed ($13,000 x g) for 2 minutes. Discard the flow-through and the collection tube.
8. Elution of RNA. Transfer the column to a clean 1.5 ml microfuge tube (not supplied with kit) and pipet 15-30 ul of DEPC-treated water (supplied with kit) into the column. Make sure to add water directly onto center of column matrix. Incubate at room temperature for 2 min. Centrifuge for 1 min at maximum speed. A second elution may be necessary if the expected yield of RNA >20 ug.
Alternatively, RNA may be eluted with a smaller volume of water. While reduced elution volume reduce the RNA yield, the concentration will be higher since more than 80% of RNA is recovered with the first elution. Pre-heating the water to 65°C before adding to column and incubating column 2 min at room temperature before centrifugation may increase yields. Elution with less than 10ul volume is not recommended.
