M465:16S rRNA gene sequencing

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(Sequencing your 16S rRNA gene)
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==Sequencing your 16S rRNA gene==
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=='''Clean up of your PCR product'''==
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e
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Before we can sesquence our bacterial 16s rRNA genes, we must remove interfering dNPTs, primers, and other small degraded DNA. We will use a column that separates DNA by size. Since the reagents and column materials in the kit we will use are proprietary, we won't know exactly what is going on at each step but, basicially, we will apply our pcr product to a column of a particular density, wash away elements too small to be trapped in it, and elute off the larger fragments of DNA (that should be ~1500bps if our pcr amplification of the 16s rRNA genes in our soil genomic DNA was successful).
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'''Notes before Starting:'''<BR>
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95% ethanol  has been added to Buffer PE before first time use (see bottle label for volume).<BR>
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All centrifuge steps are carried out at 17,900rfc (~13,000 rpm in a microcentrifuge) in a conventional tabletop microcentrifuge at room temperature.<BR>
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'''Procedure'''<BR>
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1. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction and mix.  Because our PCR reactions are 20 ul volume, we will be adding  100 ul of buffer PB to the tubes. <br> <br>
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2. Place a QIAquick column in a 2 ml collection tube. <br> <br>
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3. To bind the DNA to the column, apply the sample to the surface of the QIAquick column and centrifuge for 30-60 seconds. <br> <br>
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4. Discard the flow-through and place the QIAquick column back in the same tube. <br> <br>
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5. To wash, add 750 ul of buffer PE to the QIAquick column and centrifuge for 30-60 s.  Discard the flow-through and place the column back in the same tube. <br> <br>
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6. Centrifuge the column once more in the provided 2 ml tube to remove residual wash buffer. <br> <br>
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7. Place each QIAquick column in a clean, 1.5 mL microcentrifuge tube. <br> <br>
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8. To elute DNA, add 50 ul of buffer EB to the surface of the column - make sure to place this volume at the center of the membrane. Centrifuge the column for 1 minute. <br> <br>
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9. The purified DNA should now be at the bottom of your 1.5 mL tube. Discard the column. <br> <br>
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''IMPORTANT NOTES for using this kit: Ensure that the elution buffer (EB) is dispensed directly onto the spin column membrane for complete elution of bound DNA. The average eluate volume is 48 μl from 50 μl elution buffer volume.''<BR>
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''Elution efficiency is dependent on pH. The maximum elution efficiency is achieved
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between pH 7.0 and 8.5. Store DNA at –20°C as DNA may degrade in the absence of a buffering
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agent. ''<BR><BR>
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Make sure your pcr product is clearly labeled. Your instructor will measure the new DNA conc. using the nanodrop and post those concentrations for you in ng/μL. In the next lab you will use these amplified products for direct sequencing via the BigDye reaction. <br>

Revision as of 16:30, 19 February 2014


M465

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Clean up of your PCR product

Before we can sesquence our bacterial 16s rRNA genes, we must remove interfering dNPTs, primers, and other small degraded DNA. We will use a column that separates DNA by size. Since the reagents and column materials in the kit we will use are proprietary, we won't know exactly what is going on at each step but, basicially, we will apply our pcr product to a column of a particular density, wash away elements too small to be trapped in it, and elute off the larger fragments of DNA (that should be ~1500bps if our pcr amplification of the 16s rRNA genes in our soil genomic DNA was successful). Notes before Starting:

95% ethanol has been added to Buffer PE before first time use (see bottle label for volume).
All centrifuge steps are carried out at 17,900rfc (~13,000 rpm in a microcentrifuge) in a conventional tabletop microcentrifuge at room temperature.

Procedure
1. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction and mix. Because our PCR reactions are 20 ul volume, we will be adding 100 ul of buffer PB to the tubes.

2. Place a QIAquick column in a 2 ml collection tube.

3. To bind the DNA to the column, apply the sample to the surface of the QIAquick column and centrifuge for 30-60 seconds.

4. Discard the flow-through and place the QIAquick column back in the same tube.

5. To wash, add 750 ul of buffer PE to the QIAquick column and centrifuge for 30-60 s. Discard the flow-through and place the column back in the same tube.

6. Centrifuge the column once more in the provided 2 ml tube to remove residual wash buffer.

7. Place each QIAquick column in a clean, 1.5 mL microcentrifuge tube.

8. To elute DNA, add 50 ul of buffer EB to the surface of the column - make sure to place this volume at the center of the membrane. Centrifuge the column for 1 minute.

9. The purified DNA should now be at the bottom of your 1.5 mL tube. Discard the column.

IMPORTANT NOTES for using this kit: Ensure that the elution buffer (EB) is dispensed directly onto the spin column membrane for complete elution of bound DNA. The average eluate volume is 48 μl from 50 μl elution buffer volume.
Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5. Store DNA at –20°C as DNA may degrade in the absence of a buffering agent.


Make sure your pcr product is clearly labeled. Your instructor will measure the new DNA conc. using the nanodrop and post those concentrations for you in ng/μL. In the next lab you will use these amplified products for direct sequencing via the BigDye reaction.
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