E1 E2 E3 E4 E5 P1 P2 P3 P4 P5

Procedure

After the samples E1 E2 E3 E4 E5 P1 P2 P3 P4 P5 were qubited the information was used to determine the concentrations needed to create a mix with 100 ng of each primer

to determine the microliters needed using the following equation

100ng/ x ng/μl = x μl

These are the amounts calculated

Sample	24F	28S	IA2	ITS1
E1	2.25 μl	1.83 μl	1.08 μl	3.19 μl
E2	1.36 μl	1.17 μl	0.97 μl	3.34 μl
E3	1.89 μl	1.25 μl	0.97 μl	4.72 μl
E4	1.82 μl	1.23 μl	1.00 μl	3.24 μl
E5	1.89 μl	1.26 μl	0.94 μl	3.45 μl
P1	2.56 μl	2.24 μl	5.32 μl	3.73 μl
P2	2.52 μl	3.85 μl	7.81 μl	6.85 μl
P3	5.53 μl	2.24 μl	7.63 μl	9.85 μl
P4	2.23 μl	2.11 μl	5.67 μl	4.13 μl
P5	2.17 μl	12.02μl	10.48μl	4.85 μl

These amounts were added to the corresponding tubes so that each tube has 100 ng of each post PCR DNA.

For 24F E1-5 was taken from the 7-15-19 tube. P1-5 was taken from the 7-16-19 tube

For 28S E1-5 was taken from the 7-17-19 tube. P1-5 was taken from the 7-22-19

For IA2 E1-5 was taken from 7-23-19. P1 and P3-5 was taken from 7-24-19 P2 was from 7-29-19.

For ITS1 E1 E2 E3 and E5 was taken from 6-18-19 and E4 was taken from 6-19-19. P1-5 was taken from 6-19-19.

Notes

These tubes were placed in the -20°C (post PCR) freezer in a box titled “samples ready for sequencing”

• More primers can be added to these tubes before sequencing them.