Project: Porites Bleaching 2019

Goal

To test DNA and RNA extractions on adult Porites astreoides samples for the Porites Bleaching 2019 experiment using a modified version of this protocol.

Samples

Today I am using 6 adult P. astreoides samples that were snap frozen in the field. All of these samples are whole fragments that are stored at -80°C.

Vial #	Timepoint	Coral ID
1	A1	R8
2	A1	R36
3	A2	R29
4	A2	R28
5	A4	R11
6	A4	R20

Sample Preparation and Digestion

1. Add 0.25 mL of glass beads (0.5mm) and add 500 μl of RNA/DNA shield into each empty centrifuge tube.
2. Take samples out from -80 °C on dry ice.
3. Using sterile clippers, add 0.5mm of tissue into the centrifuge tube with beads and RNA/DNA shield.
4. Vortex at max speed for 1 minute.
5. Remove 400 μl of the supernatant and transfer to a new centrifuge tube.
6. Add 40 μl of Proteinase K digestion buffer (10:1 ratio of sample:digestion buffer), and 20 μl of Proteinase K (2:1 ratio of digestion buffer:Proteinase K) to each sample.
7. Vortex and spin down.
8. Add 460 μl of lysis buffer.
9. Continue with DNA and RNA extraction protocol

Quantify Results

Qubit

To test DNA quantity: Qubit

I used DNA and RNA broad range.

	DNA (ng/uL)	RNA (ng/uL)
Standard 1	181.19	401.73
Standard 2	19000.21	8926.01
1	7.06	26.4
2	17.8	37.6
3	23.2	22.4
4	16.0	28.6
5	16.3	34.6
6	10.7	23.6

Gel

To test DNA quality: Gel Electrophoresis

TapeStation

Sample #1:

Sample #2:

Sample #3:

Sample #4:

Sample #5:

Sample #6:

Full TapeStation report can be found here