M465:DNA Extraction and PCR

Isolating DNA from your Insects

Last time we met you cultured microbes from your wild-caught insects. However, not all microbes are culturable! Indeed, it is estimated that only one tenth of 1 percent of microbes on the planet are cultivatable. There is probably one specific microbe in your insects that is not culturable but can dramatically affect the host: Wolbachia pipientis. One of the big advantages of the molecular revolution in microbial ecology is that we can actually find, based on gene amplification, the presence of a microbe in your samples. In order to do that, we will follow the protocol below which first involves liberating the DNA in your sample, then amplifying a Wolbachia specific gene.

Isolate Genomic DNA From Your Sample
Please wear gloves during this protocol

1. Transfer 1 insect from each of your tubes of sorted insects (your groups of bugs with the same morphology) into a labeled, 1.5 mL tube.
2. To each of the tubes, add 200 uL of PBS and 20 uL of proteinase K.
3. Using a sterile pestle, grind the fly sample in the tube as well as you can. Throw the pestle in the biohazardous waste when you are done.
4. Add 200 uL of buffer AL to each 1.5 mL tube and mix by vortexing.
5. Incubate your mixture at 56C for 10 minutes (the water bath in the back).
6. Once the incubation is done, add 200 ul of ethanol (100%) to the suspension and mix by vortexing.
7. For each tube, label an individual DNeasy mini spin column in a 2mL collection tube.
8. Pipet the entire mixture into the appropriately labeled column.
9. Spin the column at 6,000 g for 1 minute. Your DNA will now have adhered to the column. Discard the flow through and place the column in a new 2 mL collection tube.

• the next steps are all washes *
  

10. Add 500 ul AW1 to the column. Centrifuge for 1 minute at 6,000 g. Discard the flow through and place the column in a new 2 mL collection tube It is extremely important that you place the column into a new tube as you will carry over the washes at each step otherwise.
11. Add 500 ul AW2 to the column and centrifuge for 3 minutes at 20,000 g (max speed). Discard the flow through and place the column in a labled, 1.5 mL tube
12. Elute the DNA from the column by adding 200 ul of buffer AE to the center of the spin column. Incubate the column for 1 minute at room temperature and spin at max speed for 1 minute.

• Your DNA is now in the liquid that came through the column!*

Finding the Wild Wolbachia

We will next attempt to amplify only Wolbachia by using a primer set specific to the bacetrium :a forward primer, wsp_F1: GTCCAATARSTGATGARGAAAC , and a reverse primer, wsp_R1: CYGCACCAAYAGYRCTRTAAA. These primers are short sequences of single stranded DNA that are complementary in sequence to areas of the wsp gene in the Wolbachia genome. The wsp gene encodes for a protein on the surface of Wolbachia. After 30 cycles of polymerase chain reaction in a thermal cycler, the result will be a pcr product containing hundreds, if not thousands, of the wsp gene, but only if your insect was infected with the bacterium.

To review how the polymerase chain reaction works and how it exponentially amplifies specific sequences of DNA, go to the following web site:
PCR animation http://www.dnalc.org/resources/animations/pcr.html

All PCR reactions require a thermal cycler to elevate and reduce the reaction temperature quickly and keep it at a specific temperature for a prescribed amount of time. There is a basic pattern to these temp. cycles, but there are differences, so you must be sure to program the cycler with the correct time and temperature for your specific amplification. Traditionally, pcr used Taq polymerase, a heat stable DNA polymerase originally found in a extremophilic bacterium, Thermus aquaticus, that lives and reproduces in boiling hot springs. We are not using Taq for our pcr but a different polymerase, Finnzyme's Phusion High-Fidelity Polymerase, a proprietary reagent that uses a novel heat-stable Pyrococcus-like enzyme. Phusion DNA Polymerase generates long templates with a greater accuracy and speed than with Taq. The error rate of Phusion DNA Polymerase in Phusion HF Buffer is determined to be 4.4 x 10-7, which is approximately 50-fold lower than that of Thermus aquaticus DNA polymerase, and 6-fold lower than that of Pyrococcus furiosus, another proof-reading DNA polymerase. Therefore, our pcr product DNA will have far fewer "mistakes" in the sequences that are replicated from template DNA. Our polymerase will also work much faster so our ~20 cycles will require less time than conventional Taq based pcr.

Protocol for PCR
1. Obtain one 0.2ml pcr tube from your instructor - you will need one for each of your DNA extractions (for each of your insect "types"). All of the ingredients listed below in the table, except the template DNA, have been added together previously and kept on ice for you in these tubes.

2. Label the tube with a fine tipped Sharpie on the side - make sure you keep track of the code name in your lab notebook. Do not use tape!

3. To each tube, you will add 4 μL of the DNA you extracted. Since your pcr tube already has 10μL master mix, 4μL DNAase free water, and 1μL of each of 2 primers, the total reaction volume for everyone will be 20μL.

It is very important to pipet these tiny volumes accurately. Use the P10 or P20 pipettes. Look at the tip after you draw up your measured volume to make sure you have liquid there.

4. Dispense the template DNA into the liquid directly, watching to make sure that the liquid has left the pipette tip.

5. Bring your tube to your instructor; they will show you where the thermal cycler is located in JH 022. Keep track of where in the PCR machine your tubes have been placed (the exact quadrant, row and column). Your instructor will start the reaction when everyone's tubes are loaded.

Component TABLE

Component	amt. in a 20 μl reaction	Final Conc.
Purified DNAase free Water	4 μL already in tube. Want to achieve total of 20 μl reaction vol. Add from 0 - 3μl	_
2x Phusion Master Mix	10 μl	1x
wspF primer	1	0.5 μMolar
wspR primer	1	0.5 μMolar
template DNA	4 μl	optimum is 100ng of DNA/reaction

The cycling program is shown below.

94°C for 2 min 37 cycles at: 94°C for 30 s 59°C for 45 s 72°C for 1 min 30 s 1 cycle at: 72°C for 10 min 4°C hold

While the 16S rRNA genes from all of the bacterial species in your DNA are being amplified in the thermal cycler, you will have about an hour to work on any other parts of your project.
After the PCR reactions are complete, you will need to complete a "Clean-Up" of your pcr products (remove the unused dNPTs, primer dimers, salts, etc. The instructions for using a kit to purify your pcr products and get them ready for sequencing next time. You will also need to set up a gel to assess the purity of your pcr product and the success of your amplification.

Storing DNA:

You can store your DNA at -20C in the freezer at the front of the lab in the box labeled M465. 

Clean Up

At the end of this lab remember to:
1) Put your DNA in the -20C freezer
2) Make sure your extra fly samples make it back to the -80C freezer (we will use them again!)
3) Wipe down your lab bench with the 70% ethanol spray bottles and paper towel
4) Dump your ice in the sink
5) Wash your hands!