Centrifuge at 3,000 x g for 5 minutes at room temperature.
Repeat Low Salt Wash Buffer wash two more times.
Resuspend oligo(dT) in 800µl Low Salt Wash Buffer using a 1-2ml serological pipette.
Transfer about 800µl of the oligo(dT) slurry to a spin column seated in the included microcentrifuge tube.
Centrifuge at 5,000 x g for 10 seconds at room temperature.
Aspirate and discard the flow-through liquid in the microcentrifuge tube.
Repeat the transfer and centrifugation steps until ALL of the oligo(dT) cellulose is in the spin column.
Elution and Precipitation of the mRNA
- Place the spin column containing the oligo(dT) into one of the included clean microcentrifuge tubes.
Heat elution buffer to 65oC in a bath (or about 10-15 seconds in a microwave).
Resuspend the oligo(dT) in 200µl Elution Buffer (heated), using the pipette tip to gently swirl the cellulose, without puncturing the underlying spin column membrane.
Centrifuge at 5,000 x g for 30 seconds at room temperature. Do not decant. Keep the eluent, it contains your mRNA.
Again resuspend the oligo(dT) in 200µl Elution Buffer (heated).
Centrifuge at 5,000 x g for 30 seconds at room temperature.
Save the combined 400µl eluent in the microcentrifuge tube.
Add 60µl of 2M sodium acetate and mix.
Add 1ml of 95% ethanol (Do not use 100% ethanol, it contains fluorescent contaminants).
Freeze at -70oC overnight.
Thaw and centrifuge at 16,000 x g for 15 minutes at 4oC.
Carefully aspirate all the ethanol from the mRNA pellet, taking care not to disturb the pellet.
Resuspend the mRNA pellet in 20µl of Elution Buffer (heated as before).
Determine the concentration of the mRNA by OD or "eyeballing" on an agarose minigel.
Store mRNA at -70oC.
The standard FastTrack protocol can be found on the Invitrogen website here
ceb 4/21/99
revised jcb 7/13/99
上一篇：Preparation of total cellular RNA 提取总RNA方法 下一篇：RNA purification using TRizol TRIZOL法提取RNA【University of Florida】
共2页: 上一页 [1] 2 下一页
