ITS2 KW-AH-ES samples
Goal
ITS2 sequencing prep on Porites astreoides and Montipora capitata samples from the Porites July Bleaching, Symbiont Integration, and Bleaching Pairs projects. 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.
The negative control was ultrapure water and the positive control was the same positive control from this ITS2 run (A12).
After the PCR amplification, the replicates in plate 2 were transferred to plate 1 and pipette mixed. Then 5uL of each sample was aliquoted back to plate 2 for the gel.
Plate 1 was stored at -20C.
20211109
Running the first dilution plate of samples with replicates
Master Mix Calculation
Plate layout
Gel Results
Today I ran a 2% gel on a 100 well gel.
20211111
Running the second plate of dilution samples and also samples that did not show a band in the previous gel. These re-do samples were diluted to 8ng/uL of DNA.
8ng/uL dilution planning
Master Mix Calculation
Plate layout
Gel Results
Today I ran a 2% gel on a 100 well gel.
20211116
Re-doing samples that failed on 20211111 with 2.5ng/uL input DNA instead of 8ng/uL.
2.5ng/uL dilution planning
I am using the DNA that was diluted to 8ng/uL.
Master Mix Calculation
Plate layout
Gel Results
Today I ran a 2% gel on a 100 well gel.
