Goal: To re-extract DNA from symbiont pellets to increase chances of amplifying symbiont ITS2 region DNA rather than host mitochondria.

Project: Porites July Bleaching 2019

Samples

All samples were snap frozen, then airbrushed with 1X PBS (Phosphate Buffer Saline). 1 mL of the homogenate was aliquoted then re-frozen at -80 °C.

Fragment_ID	Timepoint	Group	Day	Group_Day	Vial
R19	A4	Bleached	52	Bleached-52	132
R29	A4	Bleached	52	Bleached-52	204
R37	A4	Bleached	52	Bleached-52	168
R8	A4	Bleached	52	Bleached-52	210
R26	A2	Mortality	37	Mortality-37	138
R28	A2	Mortality	37	Mortality-37	72
R36	A2	Mortality	37	Mortality-37	12
R28	A4	Mortality	52	Mortality-52	144
R35	A4	Mortality	52	Mortality-52	84
R36	A4	Mortality	52	Mortality-52	120
R7	A4	Control	52	Control-52	78

DNA Extraction (performed on 20220319)

Sample Preparation and Digestion

Following the Biological fluids and cell protocol from the Zymo Quick DNA extraction kit:

1. Take samples out from -80 °C
2. Centrifuge samples for 3 minutes at 9,000 rcf and remove supernatant
3. Resuspend the pellet with 200uL of 1X PBS
4. Immediately added 200uL Biofluid Cell Buffer (red) and 20 μl of Proteinase K to each sample
5. Vortex and spin down
6. Incubate for 30 minutes at 55 °C on 1100 rpm
7. Centrifuge at 8,000 rcf for 30 seconds to remove debris
8. Transfer 350 μl of the supernatant to a new, labelled 1.5 ml centrifuge tube

DNA Extraction

1. Add equal volume (350 uL) of Genomic Binding Buffer to each tube
   • Finger flick to mix
2. Add 700 uL to the spin column
   • Centrifuge at 12,000 g (rcf) for 1 minute
3. Add 400 uL pre-wash buffer to each spin column
   • Centrifuge at 12,000 g (rcf) for 1 minute
4. Remove and discard flow through
5. Add 700 uL wash buffer to each spin column
   • Centrifuge at 12,000 g (rcf) for 1 minute
6. Remove and discard flow through
7. Add 200 uL wash buffer to each spin column
   • Centrifuge at 12,000 g (rcf) for 2 minute
8. Transfer spin columns to new 1.5 mL centrifuge tubes
9. Add 50 uL of warmed 10mM DNA Elution buffer directly to the filter in the spin column
   • Incubate at room temperature for 15 minutes
10. Centrifuge at 12,000 rcf for 1 minute
11. Add another 50 uL of warmed 10mM DNA Elution buffer directly to the filter in the spin column
    • Incubate at room temperature for 5 minutes
12. Centrifuge at 12,000 rcf for 1 minute
13. Label final tubes
14. Store labelled samples in -20 °C

Quantify Results

Qubit

DNA: Broad Range

Sample	DNA #1 (ng/uL)	DNA #2 (ng/uL)
ST 1	193.08
ST2	19841.21
12	7.88	8.2
72	21.6	22.0
78	45.2	45.8
84	21.2	21.2
120	10.0	10.5
132	39.2	39.8
138	21.4	21.8
144	22.4	22.8
168	13.1	13.2
204	19.8	20.2
210	12.8	12.9

Gel Electrophoresis

To test DNA quality: Gel Electrophoresis

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

ITS2 (performed on 20220321)

Dilution Planning

Protocol and Master Mix Planning

I am follow the Putnam Lab’s original ITS2 protocol.

Gel Electrophoresis

To test ITS2 amplification: Gel Electrophoresis

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

ITS2 (performed on 20220420)

I ran the same protocol as above except with the following thermocyler settings:

Cycles	Time	Temp
1	3 min	95°
35	30 sec	95°
	30 sec	71°
	30 sec	72°
1	2 min	72°
1	∞ min	4°	s

I decided to change the annealing temperature to 71° because this is the calculated annealing temperature for the primer with the Illumina adapters.

Gel Electrophoresis

To test ITS2 amplification: Gel Electrophoresis

• Today I did a small gel of the above protocol and ran it at 75V.