DNA/RNA extractions from coral larvae

Extractions from the coral larvae to be used for TaqSeq

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Extraction Date: January 04, 2022

Extraction notes

• Each tube contained 50 Pocillopora acuta larvae that were flash frozen in LN2
• Added 700ul of RNA/DNA shield to each tube
• The entire volume of that sample was added to a bead tube containing 0.5mm glass beads and vortexed on max speed for 30 seconds
• The tubes were briefly spun down to help remove the foam
• Pulled out 300ul from the bead tubes and added to new 1.5ml tubes. The bead tubes were placed into the -80C freezer
• Spun down samples for 3 minutes at 9000 rcf and then transfer the supernatant to new tube without disturbing the pellet
• 300ul of shield, 15ul of ProK, and 30ul of ProK digestion buffer, let sit for 2 minutes
• All spins were done for 1 minute or 2.30 minutes
• Did two washes with 700ul of wash buffer for both the DNA and RNA
• Then followed the protocol as described in protocol

Qubit

• Used Broad range dsDNA and RNA Qubit Protocol
• All samples read twice, standards only read once

DNA

Tube number	RFU	DNA 1 (ng/uL)	DNA 2 (ng/uL)	Average
Standard 1	211.59
Standard 2	22990.65
1		34.6	32.8	33.7
2		19.0	18.8	18.9
3		19.0	18.7	18.85
19		18.4	17.7	18.05
20		21.0	20.6	20.8
21		30.8	30.4	30.6
29		31.8	31.0	31.4
30		22.8	22.2	22.6
31		32.4	32.0	32.2
39		21.0	20.8	20.9
40		32.8	32.2	32.5
41		43.0	42.2	42.6
49		31.4	31.2	31.3
50		33.6	33.4	33.5
51		45.2	43.8	44.5
59		24.6	24.2	24.4
60		10.0	9.96	9.98
66		17.2	17.1	17.15
67		18.7	18.3	18.5
68		22.8	22.6	22.7
69		32.4	31.6	32.0

RNA

Tube number	RFU	RNA 1 (ng/uL)	RNA 2 (ng/uL)	Average
Standard 1	415.21
Standard 2	10815.36
1		51.2	50.0	50.7
2		33.6	33.8	33.7
3		30.0	30.8	30.4
19		26.6	25.8	26.2
20		41.2	41.0	41.1
21		36.6	36.6	36.6
29		49.8	48.8	49.3
30		35.6	35.7	35.7
31
39		45.6	45.4	45.5
40		51.0	50.8	50.9
41		49.0	48.4	48.7
49		32.0	32.2	32.1
50		36.4	36.4	36.4
51		40.0	40.0	40.0
59		38.8	38.6	38.7
60		30.0	30.0	30.0
66		23.6	23.2	23.4
67		30.2	29.8	30.0
68		35.2	34.8	35.0
69		35.8	36.2	36.0

Nanodrop

• Used ThermoScientific Nanodrop 2000
• Blanked using the water from the Zymo extraction kit

RNA

Tube number	ng/uL	260:230
1	64.1	1.60
2	44.0	1.66
3	36.4	1.45
19	36.4	1.54
20	33.2	1.73
21	35.9	1.86
29	49.6	1.69
30	47.9	1.57
31	49.8	2.09
39	52.4	1.64
40	95.6	1.04
41	59.8	1.64
49	39.5	1.56
50	39.8	1.88
51	43.6	1.91
59	36.7	1.80
60	31.1	1.66
66	25.0	1.46
67	37.2	1.36
68	36.5	1.77
69	40.0	1.71

Tape Station

• Tape station broke!

Gel

• Modified from this protocol
• Added 0.75g of agarose and 50ml of 1x TAE to flask and microwaved for 45 seconds. This makes a 1.5% gel
• Once cool enough to touch added 1ul of gel green stain
• Swirled and poured into gel mould with comb
• Once solidified, covered with 1X TAE as a running buffer
• Added 1ul of purple loading dye to each of my QC strip tube samples. I had ~9ul of DNA leftover from QC and ~8ul of RNA
• Made 2 gels, one for the DNA, one for the RNA
• Ran the gel for 50 minutes at 60 volts, the RNA gel could have used another 10 minutes to get better seperation between the bands

Addtional Notes

• 21 samples at once is way too many samples.