TITLE: PCR Preparation &amp; Gel Electrophoresis
AUTHORS: Dakota Betz

[DESCRIPTION]
How to set up a PCR reaction (Sanger sequencing only).

[BEFORE_START]
Use 75% Ethanol to clean the lab bench, tube racks, and pipettes before taking out any reagents and starting the protocol.

[GUIDELINES]
If you need to prepare primer stocks (see Primer Preparation protocol for help), do that before beginning this protocol. 

[STEPS]
SECTION: Tube and Master Mix Prep
2. Prepare your PCR tubes. To do this, use an ethanol resistant pen to label the top of each tube (use a short, 1-2 letter or number code) and the sides of each tube (repeat the short code or use a slightly longer one; also add the gene or primers that you used if there is space). Don't forget to label your negative. Be sure to note the names of each sample and the negative in your lab notebook.

Remember to thoroughly clean your workstation with 70% ethanol and 10% bleach before and after this protocol.
SECTION: Tube and Master Mix Prep
3. Make sure all reagents are completely thawed and mixed (vortex mixer) before using; these reagents should all be stored in the molecular lab freezer.
To create your PCR Master Mix, combine 8.5 µL +12.5 µL +1.0 µL +1 µL (volumes are scaled; amount for a single sample would be 8.5 uL water, 12.5 uL Taq, 1 uL fwd. primer, and 1 uL rev. primer) into a 1.6 mL eppendorf tube (lab counter, right side). 

Vortex mix the PCR Master Mix well (~15 sec). 

Be sure to note each reagent used and the date it was opened in your lab notebook.
SECTION: Scale Protocol
1. Optionally, follow along with this supplementary tutorial video as you perform PCR Preparation:
 
Double check that you have appropriately scaled this protocol. After clicking "run" above, you will need to enter your number of samples + 1-2 to account for the negative (e.g. if you are running 6 samples, scale this protocol for 7). 
SECTION: Tube and Master Mix Prep
4. Pipette 23 uL of your PCR Master Mix into each of your PCR tubes, including the negative. Close all of the tubes.
SECTION: Sample Prep &amp; Final Mixing
5. Locate each of the DNA extractions you planned to run and double check that they are in the same order as the tubes you set up in step 2. Vortex mix each of your DNA extractions for 10-15 seconds. Assure that any frozen extractions are completely thawed. Check again that the extractions are in the same order as the PCR tubes set up in step 2.
SECTION: Sample Prep &amp; Final Mixing
6. Open all PCR tubes except for the negative (remains closed). One at a time, open an extraction tube and pipette 2 uL of your DNA extraction into its corresponding PCR tube. Close the PCR tube as soon as you finish to keep track of your place.
 

SECTION: Sample Prep &amp; Final Mixing
7. Once all PCR tubes have received the appropriate amount of extraction and are closed, flip them upside down and flick a few times on either side to mix. Use the mini-tube centrifuge to spin them down briefly (5-10 sec) before placing the tubes into one of the thermal cyclers.
SECTION: Starting a Thermal Cycler Program
8. Once you've loaded your tubes and closed the cycler, note the number/name posted on the cycler in your lab notebook. On the cycler menu screen, log in to the folder you need to access (no password necessary), then choose the program you would like to use. Be sure to note the program and folder names in your lab notebook.

Once you click start, you'll see a screen that asks you to confirm the vessel being used for the reaction (tubes). Click start again, and you should see a status screen that shows that your program has begun. 
SECTION: Gel Electrophoresis &amp; ExoSAP-IT
9. Optionally, follow along with this supplementary tutorial video as you perform Gel Electrophoresis & ExoSAP-IT:
 
After your PCR program has terminated, follow the agarose gel electrophoresis instructions posted on the wall in the Ethidium Bromide Contaminated Work Station.
 

SECTION: Gel Electrophoresis &amp; ExoSAP-IT
10. Once gel electrophoresis has finished running at 120 V for 25 min, place the gel inside the UV chamber. Turn on the UV light, and take note of (in your lab notebook) which PCR samples amplified the corresponding genes you targeted.
 

SECTION: Gel Electrophoresis &amp; ExoSAP-IT
11. Place the PCR tubes that amplified correctly (thus need to be shipped out for Sanger sequencing) inside a frozen PCR tube rack, which can be found in the molecular lab freezer along with the ExoSAP-IT (inside the frozen 2-mL tube rack with a lid).
 

At the Amplified PCR Product Work Station, pipette 2 uL of ExoSAP-IT from the cap into each of the PCR tubes inside the frozen PCR tube rack. You must change pipette tips in between each sample!
 

Spin down the PCR tubes in the mini centrifuge, and insert them into an Eppendorf thermal cycler. Run the program within Jose's Favorites folder titled exosapit protocol.