DNA/RNA extractions for Porites Thermal Transplant LARVAE BATCH #1

Goal

To process DNA/RNA extractions for Porites astreoides larvae for the Thermal Transplant 2018 experiment using this protocol.

Samples

All samples were aliquots of 20 larvae released by a single colony and frozen at -80 °C.

Vial	Life Stage	Parent Coral	Date of Release
846	Larvae	R-11-B	20180710
852	Larvae	P-10-A	20180710
865	Larvae	P-19-A	20180710
868	Larvae	P-15-B	20180710
891	Larvae	R-7-A	20180711
900	Larvae	R-7-B	20180711
903	Larvae	P-4-A	20180711
906	Larvae	R-19-A	20180711
915	Larvae	R-8-A	20180711
921	Larvae	R-15-A	20180711

Sample Preparation and Digestion (LARVAE)

1. Take samples out from -80 °C.
2. Immediately add 1000 μl of RNA/DNA shield.
3. Add 100 μl of Proteinase K digestion buffer (10:1 ratio of sample:digestion buffer), and 50 μl of Proteinase K (2:1 ratio of digestion buffer:Proteinase K) to each sample.
4. Vortex and spin down.
5. Incubate for 1.5 hours at 55 °C on 1100 rpm. Check periodically to see if the samples have broken down.
6. Centrifuge at 8,000 rcf for 30 seconds to remove debris.
7. Transfer 1000 μl of the supernatant to a new, labelled 5 ml centrifuge tube.
8. Continued this protocol for RNA/DNA extractions.

Quantify Results

Qubit

DNA: Broad Range

RNA: Broad Range

Sample Type	Sample	DNA (ng/uL)	RNA (ng/uL)
	Standard 1	115.90	396.63
	Standard 2	9087.47	10288.91
Larvae	L-846	25.6	48.6
Larvae	L-852	26.4	47.0
Larvae	L-865	35.4	49.2
Larvae	L-868	21.6	24.0
Larvae	L-891	26.0	53.2
Larvae	L-900	28.4	63.8
Larvae	L-903	19.2	33.6
Larvae	L-906	55.8	51.0
Larvae	L-915	50.4	66.2
Larvae	L-921	42.6	74.6

Gel Electrophoresis

To test DNA quality: Gel Electrophoresis

• Today I did a small gel of the above protocol and ran it at 100V.

TapeStation

To test RNA quality: TapeStation

Sample #L-846:

Sample #L-852:

Sample #L-865:

Sample #L-868:

Sample #L-891:

Sample #L-900:

Sample #L-903:

Sample #L-906:

Sample #L-915:

Sample #L-921: