Testing MiFish U and E and COI on New Samples
We have been having trouble getting MiFish to work on any of our samples. When I took samples for testing the new PCR primers I filtered 3 extra liters to use for further tests with these primers.
IP-T 6 India Point Test Sample 6
GSO-T 6 Graduate School of Oceanography Beach Test Sample 6
GSO-T 7 Graduate School of Oceanography Beach Test Sample 7
- For each PCR I included a negative control
- Three Master Mixes were made with the same volumes of reagents but different primers. The total reaction volume was 25µl
- For each mix:
- 12µl Hifit Hot Start Ready Mix * 4.1 = 49.2µl
- 1.4µl forward primer * 4.1 = 5.74µl
- 1.4µl reverse primer * 4.1 = 5.74µl
- 8.2µl nuclease free water * 4.1 = 33.62µl
- 23µl of master mix was pipetted into the appropriate strip tube
- 2µl of sample DNA (or water for controls) was added to the corresponding tube
- MiFish U was run on the 65 degree program, MiFish E was run on the 70 degree program, and COI was run on the COI program
Gel
a 1.5% gel was run with GelGreen for 1 hour at 100V
Only the COI worked for the GSO samples