Goal

To stream line the protocol for DNA/RNA extractions for Porites astreoides LARVAE

Samples

Today I am using 6 larval P. astreoides samples that were snap frozen in the field. Each vial has 20 larvae. 3 were from the Thermal Transplant experiment in 2018 and 3 were from the Porites Nutrition experiment in 2019.

Vial	Sample Type	Experiment	Coral ID
12	Larvae	Porites Nutrition (2019)	P-35
27	Larvae	Porites Nutrition (2019)	P-20
161	Larvae	Porites Nutrition (2019)	P-6
550	Larvae	Thermal Transplant (2018)	R-11-A
1053	Larvae	Thermal Transplant (2018)	P-12-A
1257	Larvae	Thermal Transplant (2018)	P-19-A

Protocol description

I used a modified version of this protocol with the addition of bead beating.

Sample Preparation and Digestion

1. Take samples out from -80 °C.
2. Immediately add 500 μl of RNA/DNA shield.
3. Add 0.25 μl glass beads (0.5mm) into each sample.
4. Vortex at max speed for 30 seconds.
5. Remove 400 μl of the supernatant and transfer to a new 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	164.58	372.85
Standard 2	17530.71	8876.50
12	13.7	34.0
27	35.8	46.2
161	48.4	48.8
550	34.8	54.6
1053	39.8	40.4
1257	45.4	57.4

Gel

To test DNA quality: Gel Electrophoresis

TapeStation

Sample #12:

Sample #27:

Sample #161:

Sample #550:

Sample #1053:

Sample #1257: