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-	<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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Revision as of 16:46, 27 September 2011

{{Team:Calgary/Notebookbar|

TITLE=RNA Extraction from Pseudomonas spp.| BODY=

Reagents

1. TE buffer (10 mMTris•Cl, 1 mM EDTA, pH 8.0)containing 1 mg/ml lysozyme
2. Buffer RLT with beta-mercaptoethanol (10μL/1mL of RLT)
3. Buffer RPE with 95% ethanol (4 volumes ethanol to 1 volume ethanol)
4. RNAprotect Bacterial Reagent
5. 95% ethanol
6. RNeasy Mini Columns
7. Genome Trap Columns
8. Buffer RW1
9. Buffer RDD
10. DNase I
11. RNAse Free Water

Procedure (for Pseudomonas spp.)

1. 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
2. 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.
3. Centrifuge for 10 min at 5000g. Ensure they are balanced properly. Pellets may not be readily visible after centrifugation.
4. 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.
5. Add 100uL of TE/lysozyme buffer to each tube/extraction.
6. Vortex mix for 10s. Incubate at room temperature for 5 min on a shaker.
7. 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.
8. Add 250μL ETOH. Mix by pipetting (do not centrifuge).
9. 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.
10. 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.