DNA from samples of corals, sediments, and water collected in St. John during summer 2015 was extracted by intern George Davies using the CTAB-chloroform protocol. From preliminary PCRs, ITS2 was not successfully amplified from sediment samples. Here, I will perform a final verification that sediment samples fail to amplify ITS2, and that all negative extraction controls (i.e., blank DNA extractions) also do not amplify ITS2 (i.e., extractions are not contaminated).

PCR Setup

Reagent	Each rxn (µL)	MasterMix (µL)
H2O	18.9	699.3
10x buffer	2.5	92.5
MgCl2 @ 50 mM	1.0	37.0
dNTPs	0.25	9.25
F primer (itsDtag95 @ 10 µM)	0.5	18.5
R primer (its2rev2-bio @ 10 µM)	0.5	18.5
Taq	0.1	3.7
BSA 100x	0.25	9.25
DNA template	1

Thermocycling

3min@95°C, 35x(30s@95°C, 30s@55°C, 30s@72°C), 10min@72°C

Results

Gel 1: PCR was successful and negative extraction controls are clean

Gel 2: PCR was successful and sediment samples are remaining negative extraction controls do not amplify.

Next steps

Moving forward with library preps for coral and water samples, which are verified free of contamination by clean blank extractions. Sediment samples will not be included in library preps and sequencing because they fail to amplify.