DNA extractions for Porites Thermal Transplant ADULTS BATCH #1

Goal

To process DNA for Porites astreoides adult homogenates for the Thermal Transplant 2018 experiment using a modified version of 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	Coral
17-628	Adult Homogenate	P-10 (2017)
17-277	Adult Homogenate	R-8 (2017)
18-142	Adult Homogenate	R-8-A (2018)
18-358	Adult Homogenate	P-10-A (2018)

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

DNA: Broad Range

	DNA (ng/uL)
Standard 1	115.90
Standard 2	1087.47
17-277	10.6
17-628	24.8
18-142	37.2
18-358	12.3

Gel Electrophoresis

To test DNA quality: Gel Electrophoresis

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