gDNA extraction corals samples Mo'orea field trip 2023

Experiment context.

gDNA extraction with “Quick-DNA 96 Kit / Cat No: D3012

Protocol:

  1. Reagent preparation: Add 260ul or 1,040ul Proteinase K storage Buffer to reconstitute the lyophilized Proteinase K, 5mg (D3001-2-5) or 20mg (D3001-2-20), respectively (final concentration of 20mg/ml). Vortex to dissolve.

  2. gDNA extraction in DNA/RNA shield

    • Add 10ul PK Digestion buffer + 5ul of Proteinase K.
      For 2 plate in a time –> 2.0ml of PK Digestion Buffer + 1.0ml ProtK

    • Add 400ul og Genomic Lysis Buffer to 100ul of teh sample/shield mixture.
      For 2 plate in a time –> 400ul x 192 samples = 76.8ml

    • Mix completely by vortexing (with pipette in the plate) 4-5 times then let stand 10 minutes

    • Transfer the mixture to the wells of a Silicon-A Plate on a Collection Plate. Centrifuge at 2.500xg for 5 minutes.

    • Remove the waste in the colelction plate and move to the next step.

    • Add 200ul of DNA Pre-Wash Buffer to each well and. centrifuge at 2,500xg for 5 nminutes,
      For 2 plate in a time –> 200ul x 192 samples = 38.4ml.

    • Remove the waste in the colelction plate and move to the next step.

    • Add 300ul g-DNA Wash Buffer to each well and centrifuge at 2,500xg for 5 minutes.
      For 2 plate in a time –> 300ul x 192 samples = 57.6ml.

    • Transfer the Silicon-A Plate onto an Elution plate. Add 50ul DNA Elution Buffer (need to be heat at 70˚C) to each well and incubate during 2 minutes then centrifuge at 2,500xg for 5 minutes. For 2 plate in a time –> 50ul x 192 samples = 9.6ml.

    • Keep the Silicon-A Plate in the fridge (4˚C).

    • Cover the Elution Plate and keep it at -20˚C.

    • Nano drop 3-4 samples on each plate to control de DNA concentration.

Result:

20240925 Plate 38 and 39.

Plate Sample Well ng/ul A260/280 A260/230
Elution buffer x x 0.3 .-0.30 1.16
38 AH 180 H10 8.1 2.22 1.14
38 AH 181 H11 11.8 2.12 0.57
38 AH 182 H12 6.3 2.6 0.89
39 AH 183 A1 7.7 2.15 1.71
39 AH 197 B1 6.1 2.5 1.16
39 AH 209 C1 13.4 2.22 1.42
39 AH 25 F12 12.4 2.19 2.86
39 AH 149 G12 11.7 2.19 3.71
39 AH 162 H12 6.9 2.4 4.93

20240925 Plate 40.

Plate Sample Well ng/ul A260/280 A260/230
Elution buffer x x 0.4 1.52 0.92
40 AH 163 A1 12.8 1.9 1.87
40 AH 164 A2 10.3 2.02 3.33
40 AH 171 B1 8.6 2.11 2.23
40 AH 215 H9 1.8 3.11 0.14
40 AH 217 H10 3.7 2.94 1.03
40 AH 218 H11 1.9 6.07 0.13
40 AH 220 H12 2.6 3.83 1.16

20240926 Plate 41.

DNA extraction ecxecute with a different way on 20240925. Used Quick-DNA/RNA MiniPrep Plus Kit / Cat No.: D7003. Extraction made by Jill.

Sample ng/ul A260/280 A260/230
Elution buffer 0.1 .-0.45 0.65
AH 241 3.5 1.85 0.1
AH 237 3.1 2.23 0.08
Written on September 26, 2024