Project: Thermal Transplant Molecular

Goal

Re-doing ITS2 sequencing prep on Porites astreoides larval samples that had low quantities from the 20210302 prep and 20210309 prep. The DNA from these samples were extracted with the Zymo Quick-DNA kit and Zymo DNA/RNA mini prep kit plus.

The dilution and plate planning can be found here

Protocol

I am using a modified version of this protocol written by E. Strand.

The main modifications from this protocol were to adjust the dilutions for 2 replicates instead of 3 and each well had a total of 25 uL.

Additionally, I adjusted the volume of the Master Mix water to increase the input DNA amount accordingly.

Samples

I took the diluted samples from the dilution plate I prepped on 20210302.

Sample ID	Dilution Plate Well	Input DNA (ng)
B1-2	B1	0.2
C1-2	C1	0.2
E3-2	E3	0.2
E9-2 (POSITIVE CONTROL)	E9	0.2
B1-4	B1	0.4
C1-4	C1	0.4
E3-4	E3	0.4
E9-4 (POSITIVE CONTROL)	E9	0.4

Master Mix Calculation and Strip Tube Layout

Gel Results

Gel Electrophoresis

• I ran a small 2% agarose gel of the above protocol and ran it at 100V for 60 minutes.