- Aspirate media, wash plates with PBS 1X and aspirate off excess liquid
- Add 0.5 ml Trizol to each well in the hood, pipet up and down a few times to cover the wells, pipet the mixture into autoclaved microfuge tubes, incubate for 5’ at RT (combine the replicates into one tube)
- Add 0.2 ml Chloroform to each tube, cap tightly and shake vigorously for 15 seconds, incubate at RT for 2-3 min.
- Spin at 12,000 rpm for 15 minutes at 4C, RNA will be in the aqueous top phase.
- Transfer the aqueous phase to a fresh microfuge tube
- Add 500 ml of RNA only isopropanol to each tube, incubate at RT 10’
- Spin at 12,000 rpm for 10 minutes at 4C, look for RNA pellet
- Wash the pellet once with 1ml 75% ethanol prepared with Rnase-free water, vortex the sample to mix and spin at 7500 rpm for 5 min
- Remove the ethanol, invert tube and allow to air dry for 10-15 min
- Resuspend the pellet in 50 ml of Rnase-free water, incubate for 10’ at 50C to dissolve, store at –80
Preparation of RNA from tissues with Trizol (PJ Keller)
/0 Comments/in Molecular Biology, Protocols, RNA /by adminRNA from Tissue with Trizol (Click to Download)
*Keep tissue on dry ice until homogenization
- Add trizol to tissue, homogenize with mortar and pestle, pass through a 20G needle 2-3X to shear genomic DNA
- Incubate for 5 min at RT, keep on ice until all samples are homogenized
- Add 0.2 ml chloroform per ml Trizol, shake 15 sec, incubate 2-3 min at RT
- Spin at 12,000 x g for 10’ at 2-8C
- Transfer the clear aqueous phase to a new tube, add 0.5 ml isopropyl alcohol
- Incubate at RT 10’, centrifuge at 12,000 x g for 10’ at 2-8C
- Remove the supernatant, wash the pellet with 1 ml 75% EtOH, vortex
- Spin down pellet at 7500 x g for 5’ at 2-8C
- Air dry the RNA pellet 5-10’, resuspend in 110 ml RNase-free H20, remove 10 ml as an aliquot for pre-cleanup analysis
Use Qiagen RNeasy kit to clean up RNA prep (note-may want to add on-column DNAase digestion, see protocol with kit) - Make up buffer RLT (3.5 ml) by adding 35 ml BME to 3.5 ml RLT
- Add 350 ml RLT to 100 ml Trizol RNA sample, mix
- Add 250 ml 100% EtOH to the diluted RNA, mix
- Add the sample to a mini column placed in a 2 ml collection tube
- Spin 15s at 10,000 rpm
- Transfer the column to a new collection tube, add 500 ml RPE, spin 15s
- Discard the flow through, wash again with 500 ml RPE, spin 2’
- Discard Flow through, spin again 1’
- Elute into a 1.5 ml collection tube with 50 ml RNase-free water, spin 1’
- Dilute a fraction at 1:100 in 10 mM Tris pH 8 and measure concentration
Aliquot small fractions of RNA and store at -80
RNA preparation from plates with Trizol (PJ Keller)
/0 Comments/in Molecular Biology, Protocols, RNA /by adminRNA Prep Trizol (Click to Download)
Contact Us
Charlotte Kuperwasser, PhD
Associate Professor of Developmental, Molecular & Chemical Biology
Tufts University School of Medicine
Investigator
Molecular Oncology Research Institute (MORI)
Tufts Medical Center
Boston, MA 02111
(617) 636-3828
Charlotte.Kuperwasser@Tufts.edu
