DNA extractions for Porites Thermal Transplant ADULTS BATCH #3

Goal

To process DNA for Porites astreoides adult homogenates for the Thermal Transplant 2018 experiment using this protocol.

Samples

All samples were snap frozen, then airbrushed with filtered seawater. 0.5 mL of the homogenate was aliquoted then re-frozen at -80 °C.

Vial	Sample Type	Year	Coral ID
17-214	Adult Homogenate	2017	R-4
17-232	Adult Homogenate	2017	R-11
17-250	Adult Homogenate	2017	R-18
17-538	Adult Homogenate	2017	P-4
17-547	Adult Homogenate	2017	P-14
18-418	Adult Homogenate	2018	R-9-A
18-406	Adult Homogenate	2018	R-15-A
18-394	Adult Homogenate	2018	P-9-B
18-382	Adult Homogenate	2018	R-4-B
18-370	Adult Homogenate	2018	P-15-A

Sample Preparation and Digestion

• Following the Biological fluids and cell protocol

1. Take samples out from -80 °C.
2. Immediately added 500uL Biofluid Cell Buffer (red) and 50 μl of Proteinase K to each sample.
3. Vortex and spin down.
4. Incubate for 30 minutes at 55 °C on 1100 rpm.
5. Centrifuge at 8,000 rcf for 30 seconds to remove debris.
6. Transfer 1000 μl of the supernatant to a new, labelled 5 ml centrifuge tube.

DNA Extraction

1. Add equal volume (1000 uL) of Genomic Binding Buffer to each 5 ml tube
   • Finger flick to mix
2. Add 700 uL of the 5 mL tube to the spin column
   • Centrifuge at 12,000 g (rcf) for 1 minute
3. Transfer spin column to a new column (discard previous column)
4. Repeat steps 2-3 until the liquid is gone (3x)
5. Add 400 uL pre-wash buffer to each spin column
   • Centrifuge at 12,000 g (rcf) for 1 minute
6. Remove and discard flow through
7. Add 700 uL pre-wash buffer to each spin column
   • Centrifuge at 12,000 g (rcf) for 1 minute
8. Remove and discard flow through
9. Add 200 uL wash buffer to each spin column
   • Centrifuge at 12,000 g (rcf) for 2 minute
10. Transfer spin columns to new 1.5 mL centrifuge tubes
11. Add 50 uL of warmed 10mM Tris HCl directly to the filter in the spin column
    • Incubate at room temperature for 15 minutes
12. Centrifuge at 12,000 rcf for 1 minute
13. Add another 50 uL of warmed 10mM Tris HCl directly to the filter in the spin column
    • Incubate at room temperature for 5 minutes
14. Centrifuge at 12,000 rcf for 1 minute
15. Label final tubes
16. Aliquot 10 uL into labelled PCR tubes for QC
17. Store labelled samples in -20 °C

Quantify Results

Qubit

To test DNA quantity: Qubit

DNA: Broad Range

	DNA (ng/uL)
Standard 1	164.58
Standard 2	17530.71
17-214	39.0
17-232	10.8
17-250	30.0
17-538	31.8
17-547	48.8
18-418	34.0
18-406	26.4
18-394	9.26
18-382	18.5
18-370	19.3

Gel Electrophoresis

To test DNA quality: Gel Electrophoresis

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