Salmon is back and running!

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Chelex Extraction and PCR      Sunday 02.26.2017

Hi everyone,

We decided to check out protocols by doing independent runs on the weekends. Here’s my update.

Salmon DNA by Chelex Extraction

  1. Measure a .6mg piece of salmon from freezer
  2. Added to 300ul of 5% Chelex and vortexed for 30sec
  3. Incubated at 95C for 6min
  4. Vortexed
  5. Incubated at 95C for 6 min
  6. Centrifuged at 13000 for 5 min
  7. Remove and keep supernatant; discard pellet

PCR

1 rxn 36(+2 extra) rxn
ddH2O 38 ul 1444 ul
PCR Master Mix 6x (Cell&Gene) 10 ul 380 ul
Primers F&R (10ug/ml) 1 ul 38 ul (19 ul each primer)
Template DNA 1 ul 38 ul

 

PCR was run on “Sleepy” with a annealing temperature gradient of 42C-52C for columns 1-12.

Program: Main -> [B start/stop] Choose Directory -> [Down arrow -9. SoundBio, D enter] Find program -> [A list, COXX] confirm program -> [D enter] start program [D start]

 

Thermocycler – Biometra Tgradient Temp Time
Initial Denaturation 95 30(s)
Denature 95 15(s)
30 Cycles
Annealing 42-52 15(s)
Extension 72 60(s)
Final Extension 72 5(m)
Hold 4

 

Tested 3 different primer sets for the COXX gene.

  • Primer set 2 = purple
  • Primer set 3 = yellow
  • Primer set 6 = blue

Tested serial dilutions using ddH2O.

Final key:

  1. Primer set 2 – full conc.
  2. Primer set 2 – 1/10th dilution
  3. Primer set 3 – full conc.
  4. Primer set 3 – 1/10th dilution
  5. Primer set 3 – 1/100th dilution
  6. Primer set 6 – full conc.
  7. Primer set 6 – 1/10th dilution
  8. Primer set 6 – 1/100th dilution

PCR machine was set to run overnight, then moved to 4C fridge.