Goal

Attempting the lipid assay using the unmodified protocol from Bove_Baumann_96well_Protocol on multiple different coral species and host/symbiont fractions.

The working Putnam Lab Lipid protocol is here

Samples and Sample Preparation

For this protocol, I used airbrushed homogenates of Astrangia poculata (Proj; Astrangia Nutrition), Porites astreoides (Proj: Porites Nutrition 2019), Montipora capitata (Proj: Holobiont Integration), and Pocillopora acuta (Proj: Holobiont Integration). These were extra coral homogenates that were frozen is 50mL falcon tubes.

Samples were taken out of the -80C freezer to thaw. Once thawed, 1 mL aliquots were put into the Holobiont and Symbiont tubes. The symbiont tubes were centrifuged for 3 minutes at 15000 rcf. The supernatant was transferred to the associated ‘coral’ tube and the pellet was resuspended in 1X PBS (phosphate buffer saline).

Vial	Fragment ID	Species	Fraction
1	1246	A. poculata (apo)	Coral
2	1246	A. poculata (apo)	Symbiont
3	2012	A. poculata (sym)	Coral
4	2012	A. poculata (sym)	Symbiont
5	P22-A2	P. astreoides	Coral
6	P22-A2	P. astreoides	Symbiont
7	M1822	M. capitata	Coral
8	M1822	M. capitata	Symbiont
9	P1170	P. acuta	Coral
10	P1170	P. acuta	Symbiont

Results

Notes

• After centrifugation in cholorform and methanol:
  • P. astreoides coral fraction in the chloroform was still green
  • Large “cellular debris” layer in some samples (see below)
• Samples 1 and 9 may have a bit of the cellular debris layer in the well due to pipette error (this actually resulted in positive readings I think)
• No samples in wells A6, A9, D5, F1 due to pipette error.

Below are photos of the samples after the shaker plate and centrifugation with chloroform and methanol.