Team:Calgary/Notebook/Protocols/Process2
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<br> | <br> | ||
- | <h4> Procedure </h4> | + | <h4> Procedure (for <i>Pseudomonas spp.</i>)</h4> |
+ | <ol> | ||
+ | <li>Calculate the OD600 of each culture used for RNA extraction. Transfer 2x10^8 cells into a new separate microfuge tubes for each RNA extraction.Calculate the amount of cultures to be dispensed using 1x10^9 cells/mL= ~1 OD600</li> | ||
+ | <li>Add 2 volumes of RNA protect to each tube (corresponding to the volume of cells that contain 2x10^8 cells), vortex for 5 sec. and incubate at room temperature for 5 minutes.</li> | ||
+ | <li>Centrifuge for 10 min at 5000g. Ensure they are balanced properly. Pellets may not be readily visible after centrifugation. </li> | ||
+ | <li>Decant the supernatant and remove residual liquid from the tube by dabbing inverted tube with a paper towel and leave inverted on the towel for ~10s.</li> | ||
+ | <li>Add 100uL of TE/lysozyme buffer to each tube/extraction.</li> | ||
+ | <li>Vortex mix for 10s. Incubate at room temperature for 5 min on a shaker.</li> | ||
+ | <li>Add 350μL of Buffer RLT/β-mercaptoethanol solution and vortex vigorously. If there is any particulate in the tube, pellet it by centrifugation and keep the supernatant.</li> | ||
+ | <li>Add 250μL ETOH. Mix by pipetting (do not centrifuge).</li> | ||
+ | <li>Transfer up to 700μL of lysate (including precipitate) to an RNeasy Mini spin column placed in a 2mL collection tube. Centrifuge 15s at ~8000 x g. Discard flow-through.</li> | ||
+ | <li> Treat on RNeasy column with DNAse, or alternatively pass through a genome trap column and collect the flow through. Genomic DNA will be retained in the column whereas RNA will pass through.</li> | ||
+ | |||
+ | </ol> | ||
+ | <br> |
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TITLE=RNA Extraction from Pseudomonas spp.|
BODY=
Reagents
- TE buffer (10 mMTris•Cl, 1 mM EDTA, pH 8.0)containing 1 mg/ml lysozyme
- Buffer RLT with beta-mercaptoethanol (10μL/1mL of RLT)
- Buffer RPE with 95% ethanol (4 volumes ethanol to 1 volume ethanol)
- RNAprotect Bacterial Reagent
- 95% ethanol
- RNeasy Mini Columns
- Genome Trap Columns
- Buffer RW1
- Buffer RDD
- DNase I
- RNAse Free Water
Procedure (for Pseudomonas spp.)
- Calculate the OD600 of each culture used for RNA extraction. Transfer 2x10^8 cells into a new separate microfuge tubes for each RNA extraction.Calculate the amount of cultures to be dispensed using 1x10^9 cells/mL= ~1 OD600
- Add 2 volumes of RNA protect to each tube (corresponding to the volume of cells that contain 2x10^8 cells), vortex for 5 sec. and incubate at room temperature for 5 minutes.
- Centrifuge for 10 min at 5000g. Ensure they are balanced properly. Pellets may not be readily visible after centrifugation.
- Decant the supernatant and remove residual liquid from the tube by dabbing inverted tube with a paper towel and leave inverted on the towel for ~10s.
- Add 100uL of TE/lysozyme buffer to each tube/extraction.
- Vortex mix for 10s. Incubate at room temperature for 5 min on a shaker.
- Add 350μL of Buffer RLT/β-mercaptoethanol solution and vortex vigorously. If there is any particulate in the tube, pellet it by centrifugation and keep the supernatant.
- Add 250μL ETOH. Mix by pipetting (do not centrifuge).
- Transfer up to 700μL of lysate (including precipitate) to an RNeasy Mini spin column placed in a 2mL collection tube. Centrifuge 15s at ~8000 x g. Discard flow-through.
- Treat on RNeasy column with DNAse, or alternatively pass through a genome trap column and collect the flow through. Genomic DNA will be retained in the column whereas RNA will pass through.