DNA/RNA extractions for Porites Thermal Transplant Trial #4 - Adults and Larvae

Goal

To stream line the protocol for DNA/RNA extractions for Porites astreoides ADULTS and LARVAE for the Thermal Transplant 2018 experiment.

ADULTS - I am trying to increase the concentration of the DNA and RNA without composmising integrity. I will attempt increasing the input volume of sample and follow Trial 1 ADULT.

LARVAE - I am trying to increase the quality of DNA and RNA of the larvae by decreasing the incubation (digestion) period from Trial 1 LARVAE.

Samples

Today I am using:

• 2 adult P. astreoides samples that were already chipped (~0.5mm diameter) and placed in 500 uL of DNA/RNA Shield.
  • #9 and #61
• 2 larval P. astreoides samples that were snap frozen in the field. Each vial has 20 larvae.
  • #871 and #1087

Sample Preparation and Digestion (ADULTS)

1. Take samples out from -80 °C.
2. Add 500 μl of RNA/DNA shield to make the total volume 1 mL
3. Add ~0.25 ml of 0.5mm glass beads to each sample
4. Vortex for 2 minutes. Leave the settings on and on max power.
5. Remove 700 μl of the supernatant from the bead tube and place in a new 1.5 microcentrifuge tube labeled on the side with the extraction sample number and today’s date. Label the cap of the microcentrifuge tube with the sample number.
6. Add another 500 μl of RNA/DNA shield to the bead tube.
7. Gently mix by inversion.
8. Remove 500 μl of the supernatant from the bead tube and transfer to the remainder of the supernatant.
9. Add 120 μl of Proteinase K digestion buffer (10:1 ratio of sample:digestion buffer), and 60 μl of Proteinase K (2:1 ratio of digestion buffer:Proteinase K) to the 5 mL centrifuge tube.
10. Vortex and centrifuge at 16,000 rcf for 30 seconds to remove debris.
11. Transfer 1200 μl of the supernatant to a new, labelled 5 ml centrifuge tube.
12. Vortex and spin down all tubes.

Sample Preparation and Digestion (LARVAE)

1. Take samples out from -80 °C.
2. Immediately add 300 μl of RNA/DNA shield.
3. Add 30 μl of Proteinase K digestion buffer (10:1 ratio of sample:digestion buffer), and 15 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 16,000 rcf for 30 seconds to remove debris.
7. Transfer 300 μl of the supernatant to a new, labelled 1.5 ml centrifuge tube.

Quantify Results

Qubit

4 samples + 2 standards + 1 error = 7

DNA Broad Range

199 µl Buffer x 7 = 1393 µl Buffer 1 µl Reagent x 7 = 7 µl Reagent

RNA Broad Range

199 µl Buffer x 7 = 1393 µl Buffer 1 µl Reagent x 7 = 7 µl Reagent

	S1	S2	9 (ADULT)	61 (ADULT)	871 (LARVAE)	1087 (LARVAE)
DNA	152.72	21489.09	5.14	10.1	22.4	75.8
RNA	394.33	10395.22	LOW	24.0	29.6	56.4

TapeStation

Sample #9:

Sample #61:

Sample #871:

Sample #1087:

Gel