mtORF PCR of Pocillopora spp for Caroline Dube

Loosely based off of this protocol.

Diluted the genomic data 1:10 with PCR grade water in strip tubes to help reduce any inhibitors that might be in the sample. Not worried about final concentration.

• First PCR Samples:
  • Positive control- E5 123
  • Negative control
  • 61
  • 68
  • 76
  • 77
  • 78
  • 87
  • 91
  • 108
  • 113
  • 371
  • 376
  • 394

Made 15 total PCR reactions

1. 249.9ul of Phusion High-Fidelity PCR Master Mix
2. 219.9ul of Ultrapure PCR grade water
3. 6.45ul of 10uM FatP6 primer
4. 6.45ul of 10uM RORF primer

• 32ul of MM into each strip tube and 1ul of the diluted gDNA
• Spun down tubes briefly

Gel

• Modified from this protocol
• Added 0.50g of agarose and 50ml of 1x TAE to flask and microwaved for 45 seconds. This makes a 1.0% gel
• Once cool enough to touch added 5ul of gel red stain
• Swirled and poured into gel mould with comb
• Once solidified, covered with 1X TAE as a running buffer
• Added 1ul of purple loading dye to a piece of parafilm and then added 3ul of PCR product.
• Gel ran for 30 minutes at 80V

Ethanol precipitation

1. Add 3ul of 3M sodium acetate to each PCR reaction (1/10th the total volume)
2. Add 99ul of ice cold 100% ethanol to each tube (3x the total volume)
3. Transfer entire volume to a new labeled 1.5ml tube
4. Place tubes in -80 freezer for about 30 minutes
5. Spin for 30 minutes at 12,000rpm. Make sure all the tubes are facing the same way. The DNA will pellet on the back wall
6. Pipette off the liquid without disturbing the pellet. It will most likely be invisible.
7. Add 200ul of ice cold 70% Ethanol
8. Spin for 5 minutes at 12,000rpm. This is to wash the pellet. Again make sure each tube is facing the same direction
9. Pipette off the ethanol without disturbing the pellet
10. Let dry at room temperature with the caps open for about 15 minutes or until all ethanol has evaporated
11. Add 30ul of either Tris or water to the pellet and pipette up and down to resuspend

Nanodrop

DNA	ng/ul	260/280
61	145.7	1.72
68	162.3	1.70
76	141.2	1.71
77	133.1	1.72
78	111.3	1.70
87	208.8	1.66
91	110.2	1.70
108	151.7	1.70
113	149.0	1.72
371	102.4	1.69
376	107.1	1.68
394	148.3	1.72

Sequencing

• had to dilute the DNA 1:10 so it would have a low enough concentration to set up the samples up for sequencing. I did not re-nanodrop the samples after diluting
• Set up samples in just the forward direction first, then set up the reverse direction once I made the sure quality looked good