Team:Calgary/Notebook/Protocols/Process2

From 2011.igem.org

(Difference between revisions)
 
(11 intermediate revisions not shown)
Line 5: Line 5:
BODY=<html>
BODY=<html>
-
<br>
+
<p>This protocol isolates total RNA from <i>Pseudomonas spp.</i> (or gram-negative bacteria) for use in qRT-PCR. The protocol can be obtained from Qiagen's RNeasy Kit and Bacterial RNAprotect Kit manuals.</p>
-
<h4> Reagents </h4>
+
<h4> Reagents and Materials</h4>
-
<ol>
+
 
-
<li>TE buffer (10 mMTris•Cl, 1 mM EDTA, pH 8.0)containing 1 mg/ml lysozyme</li>
+
<li>TE buffer (10 mM Tris•HCl, 1 mM EDTA, pH 8.0) containing 1 mg/mL lysozyme</li>
<li>Buffer RLT with beta-mercaptoethanol (10μL/1mL of RLT)</li>
<li>Buffer RLT with beta-mercaptoethanol (10μL/1mL of RLT)</li>
<li>Buffer RPE with 95% ethanol (4 volumes ethanol to 1 volume ethanol)</li>
<li>Buffer RPE with 95% ethanol (4 volumes ethanol to 1 volume ethanol)</li>
<li>RNAprotect Bacterial Reagent </li>
<li>RNAprotect Bacterial Reagent </li>
 +
<li>Proteinase K</li>
<li>95% ethanol</li>
<li>95% ethanol</li>
<li>RNeasy Mini Columns</li>
<li>RNeasy Mini Columns</li>
Line 19: Line 20:
<li>DNase I</li>
<li>DNase I</li>
<li>RNAse Free Water</li>
<li>RNAse Free Water</li>
-
</ol>
+
 
<br>
<br>
<h4> Procedure (for <i>Pseudomonas spp.</i>)</h4>
<h4> Procedure (for <i>Pseudomonas spp.</i>)</h4>
<ol>
<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>Calculate the OD<sub>600</sub> 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 OD<sub>600</sub></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>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>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>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>Add 100μL of TE/lysozyme buffer and 5 µL of Proteinase K to each tube/extraction.</li>
-
<li>Vortex mix for 10s. Incubate at room temperature for 5 min on a shaker.</li>
+
<li>Vortex mix for 10s. Incubate at room temperature for 10 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 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>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>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>
+
<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. For DNAse treatment procedure, use the following steps in the order listed.</li>
-
 
+
<ul>Add 350μL of Buffer RW1 to minispin column and centrifuge for 15s at 8000g to wash the spin column membrane.  Discard flow-through.</ul>
 +
<ul>Add 10μL or DNase I stock solution to 70μL or Buffer RDD.  Mix by gently inverting the tube and centrifuge briefly to collect residual liquid from the sides of the tube</ul>
 +
<ul>Add the DNase I incubation mix (80μL) directly to the RNeasy spin column membrane, and incubate at room temperature for 15 min.</ul>
 +
<ul>Add 350μL or Buffer RW1 to the RNeasy spin column, wait for 5 min, and then centrifuge for 15s at 8000g.  Discard the flow-through and collection tube.</ul>
 +
<li>Put RNeasy column in new 2mL collection tube.  Add 500μL RPE to the mini-spin column.  Centrifuge again for 15s at 8000g.</li>
 +
<li>Add 500μL RPE to the mini-spin column.  Centrifuge for 2min. at 8000g for a final wash.</li>
 +
<li><b><i>(Optional)</b></i> Place column in a clean and empty collection tube, and spin at top speed for 1 min.</li>
 +
<li>Place the RNeasy mini spin column in a new 1.5mL collection tube.  Add 30-50μL of RNAse-free water directly to the spin column membrane.  Centrifuge 1 min at 8000g to elute the RNA.</li>
 +
<li>For maximum yield, add another 30μL of RNAse-free water (or eluate from previous step) to the column and centrifuge into the same collection tube.</li>
</ol>
</ol>
<br>
<br>
 +
</html>}}

Latest revision as of 04:03, 29 October 2011


RNA Extraction from Pseudomonas spp.

This protocol isolates total RNA from Pseudomonas spp. (or gram-negative bacteria) for use in qRT-PCR. The protocol can be obtained from Qiagen's RNeasy Kit and Bacterial RNAprotect Kit manuals.

Reagents and Materials

  • TE buffer (10 mM Tris•HCl, 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
  • Proteinase K
  • 95% ethanol
  • RNeasy Mini Columns
  • Genome Trap Columns
  • Buffer RW1
  • Buffer RDD
  • DNase I
  • 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 100μL of TE/lysozyme buffer and 5 µL of Proteinase K to each tube/extraction.
    6. Vortex mix for 10s. Incubate at room temperature for 10 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. For DNAse treatment procedure, use the following steps in the order listed.
        11. Add 350μL of Buffer RW1 to minispin column and centrifuge for 15s at 8000g to wash the spin column membrane. Discard flow-through.
        Add 10μL or DNase I stock solution to 70μL or Buffer RDD. Mix by gently inverting the tube and centrifuge briefly to collect residual liquid from the sides of the tube
        Add the DNase I incubation mix (80μL) directly to the RNeasy spin column membrane, and incubate at room temperature for 15 min.
        Add 350μL or Buffer RW1 to the RNeasy spin column, wait for 5 min, and then centrifuge for 15s at 8000g. Discard the flow-through and collection tube.
    11. Put RNeasy column in new 2mL collection tube. Add 500μL RPE to the mini-spin column. Centrifuge again for 15s at 8000g.
    12. Add 500μL RPE to the mini-spin column. Centrifuge for 2min. at 8000g for a final wash.
    13. (Optional) Place column in a clean and empty collection tube, and spin at top speed for 1 min.
    14. Place the RNeasy mini spin column in a new 1.5mL collection tube. Add 30-50μL of RNAse-free water directly to the spin column membrane. Centrifuge 1 min at 8000g to elute the RNA.
    15. For maximum yield, add another 30μL of RNAse-free water (or eluate from previous step) to the column and centrifuge into the same collection tube.

    Retrieved from "http://2011.igem.org/Team:Calgary/Notebook/Protocols/Process2"