Visited Point Reyes (about 45 miles north of San Francisco) on 9/20/2017 to collect samples of the purple-flowered variant of Bush Lupin (Lupinus excubitus) for genetic sequencing and population comparison.

Protocol:

1. Recorded sample info and labeled 1.5 ml screw cap tubes with the following:
   1. JPCA – Escolar
   2. JPCB – Yellowtail
   3. JPCC – Black Snapper
   4. JPCD – Albacore Tuna
   5. 
2. Snipped 2-10 mg samples of each fish species (weighed the first one and approximated the rest), wiped blade with ethanol between each change of species
3. Added 100 microliters of Extraction Solution and 25 microliters of Tissue Preparation to labeled tubes (solutions from Sigma REDExtract-N-Amp Tissue PCR Kit)
4. Added samples to each tube
5. Incorrectly incubated tubes at 95 degrees Celsius for 10 mins
6. Removed tubes from heating block, allowed them to cool for 5 minutes
7. Added 100 microliters of Neutralizing Solution from same kit
8. Vortexed for 10 seconds
9. Put samples on ice
10. Made 10x dilution of gDNA- 18 microliters diH2O + 2 microliters of gDNA
11. PCR Reagents for each tube (14 Reaction overall Master Mix)
    1.  Note: Pipette was somewhat wonky
    2. PCR Quality Water – 6.4 µl
    3. REDExtract-N-Amp PCR rx mix – 10 µl
    4. Forward Primer – 11.2 µl
    5. Reverse Primer – 11.2µl
12. added 2 µl Diluted Tissue Extract to each labeled PCR tube
13. Put into thermocycler with following settings
    1. 94 C – 4 minutes (initial denaturation)
    2. 30 Cycles of
       1. 94 C for 30 sec (denaturing)
       2. 52 C for 40 sec (annealing)
       3. 72 C for 1 min (extension)
    3. 72 C hold for 10 min (final extension)
    4. 10 C hold indefinitely