M465:16S rRNA gene sequencing

Today we will be cleaning up your PCR product, quantifying the DNA, and performing a big-dye sequencing reaction. This kind of sequencing uses Sanger chemistry (more information on that method can be found here and will be covered in class.

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!

Measuring the Quantity of DNA in your PCR product

We will measure the quantity of DNA in your cleaned up PCR reactions using the Synergy H1 spectrophotometer and a Take3 plate. The plate allows us to spot a very small quantity of your DNA (2 ul) on the surface of a glass slide and the spectrophotometer will measure the quantity for us. The Take3 plate can measure 16 samples at once so three of you can measure your PCR reactions from each of your 5 isolates each time.

The spectrophotometer will measure the DNA quantity by taking Absorbance values at A_260nm. You can watch this video on using the Take3 plates here

Using the Take3 Plates with the Synergy H1 Spectrophotometer

Each column of the Take3 plate contains 8 spots where you will place your sample. On the top left spot, you will spot 2 uL of a "blank", in our case, the EB buffer used to elute the DNA from the column.
For each sample you want to measure, you will use the P20 micropipette to spot 2 uL of your sample onto the surface of the Take3 plate. Once the columns are filled, slowly and carefully depress the top of the plate so that the two glass slides are touching. *Do this in a controlled fashion as these magnetic plates can crack the slides*
Using the Synergy H1 software, open the "Take 3 nucleic acid quantification" experiment. Select the upper left spot as your blank and the rest of the wells as your sample. Make sure that you are measuring the DNA concentration (check the drop-down menu at the top of the experiment page). After your acquisition is done, press "accept" to transfer the data to Excel and save your results.

Clean Up When the last sample was been measured, clean the Take3 plates with a kim-wipe. No solvent is necessary.

Performing a Big-Dye Reaction

Your instructors will provide you with PCR tubes, preloaded with the sequencing reaction components. All you need to do is add your purified, PCR fragment. For each of your amplified 16S rRNA gene products, you will perform the following:

• All of the steps below should be performed on ice!*

1) Label a 200 ul sequencing reaction tube with your isolate number and your initials.
2) Using the P20, take 4 ul of your *clean*, and quantified PCR product, and add this to the sequencing reaction tube. Be careful as to not introduce bubbles.
3) Using the centrifuge with the 200 ul tube adapter, spin down your sequencing reactions.
4) Load your sequencing reactions into the PCR machine, marking the location of your tubes on the 96-well template to the right of the machine.

The cycling program for a BigDye sequencing reaction is shown below.

Thermal Cycler Program:
3 step program

Cycle Step	Temperature	Time	# of Cycles
Initial Denaturation	95C	1 min.	1
Denaturation Annealing Extension	95C 50C 60C	10 sec 5 min 4 min	30

Next time we meet, we will have your sequences and you will begin the bioinformatic analysis!
Note: Each sequencing reaction contains 1 uL of 27F sequencing primer (at 3.2 mM concentration), 1 uL of distilled, sterile water, and 4 uL of Big-Dye reaction mix.