Difference between revisions of "Preparation of RNA Samples from Mouse Tissues"
From Bridges Lab Protocols
Davebridges (Talk | contribs) (updated for purelink kits.) |
Davebridges (Talk | contribs) (added ethanol to materials and prepared TRIzol tubes first) |
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*Chloroform (in solvent cabinet) | *Chloroform (in solvent cabinet) | ||
*Label tubes, for each sample need 3 eppies, at least one of which is 2 mL and one of which is a recovery tube from the PureLink kit. | *Label tubes, for each sample need 3 eppies, at least one of which is 2 mL and one of which is a recovery tube from the PureLink kit. | ||
| − | + | *70% Ethanol make with RNAase free water and 100% Ethanol. | |
==Protocol== | ==Protocol== | ||
| − | #Cut ~50-100 mg of tissue. If tissue is in RNAlater, cut at room temperature. If tissue is frozen cut on dry ice. Weigh into a fresh 2mL eppendorf tube and keep on | + | #Add 1 mL TRIzol reagent to each 2 mL tube. |
| − | + | #Cut ~50-100 mg of tissue. If tissue is in RNAlater, cut at room temperature. If tissue is frozen cut on dry ice. Weigh into a fresh 2mL eppendorf tube and keep on ice. | |
#Using tissue grinder, homogenize tissue for ~30s until homogeneous (no clumps). | #Using tissue grinder, homogenize tissue for ~30s until homogeneous (no clumps). | ||
#Incubate 5 minutes at room temperature. | #Incubate 5 minutes at room temperature. | ||
Revision as of 17:00, 11 January 2012
Materials
- PureLink RNA Mini Kit (Invitrogen cat#12183-018A)
- Mouse Tissue (50-100 mg, about a 3mm cube)
- TRIZol (Invitrogen cat# 12183-555)
- Chloroform (in solvent cabinet)
- Label tubes, for each sample need 3 eppies, at least one of which is 2 mL and one of which is a recovery tube from the PureLink kit.
- 70% Ethanol make with RNAase free water and 100% Ethanol.
Protocol
- Add 1 mL TRIzol reagent to each 2 mL tube.
- Cut ~50-100 mg of tissue. If tissue is in RNAlater, cut at room temperature. If tissue is frozen cut on dry ice. Weigh into a fresh 2mL eppendorf tube and keep on ice.
- Using tissue grinder, homogenize tissue for ~30s until homogeneous (no clumps).
- Incubate 5 minutes at room temperature.
- Add 200 uL Chloroform and shake vigourously by hand for 15s. Do not vortex.
- Incubate at room temperature for 2-3 minutes.
- Centrifuge in a cold eppendorf centrifuge on maximum for 15 minutes. Sample will separate into a lower, red chloroform phase, an interphase and a colorless upper phase which contains the RNA.
- Transfer 600 uL of the upper phase to a fresh tube.
- Add 600 uL of 70% ethanol and mix by vortexing.
- Remove 700 uL of mixture (invert briefly to mix before taking sample) and add to a spin column in a collection tube.
- Spin 15s on max (press button). Discard flow through. Add remaining sample and respin.
- Add 700 uL Wash Buffer I to spin column.
- Spin 15s on max. Discard flow through and the collection tube. Get a new collection tube.
- Add 500 uL Wash Buffer II (make sure ethanol was added to the wash buffer) to the spin cartridge.
- Spin 15s on max. Discard the flow through and replace the collection tube.
- Repeat wash by adding 500 uL Wash Buffer II to the spin cartridge.
- Spin 15s on max. Discard the flow through and keep the same collection tube.
- Spin 1 min on max to dry the cartridge. Discard the collection tube and place into a clean recovery tube. Add 100 uL RNAase free water to the center of each tube. This can be adjusted to between 30 and 300 uL elution buffer if necessary.
- Incubate at room temperature for 1min.
- Spin 2 min on max to get purified RNA.
- Quantify the RNA using the nanodrop.
