Test MiFish PCRs again
Testing PCRs again with new primer stocks
Made new diluted stocks of MiFish E and U by diluting stock with 90ul low TE buffer and 10ul of primer
Samples Tested:
- eDNA test clean
- Test 3
- Test 4
- K 0.4 1 Liter
- H 12 1 Liter
- J 12 1 Liter
- M 0.4 1 Liter
Samples K, H, J, and M are Diana’s, the first three were filtered from GSO beach
Testing MiFish U at 65 degree PCR program and testing Mifish E at 70 degree PCR program
Made 2 PCR master mixes for 16 samples total:
12ul KAPA HiFi Hot Start Ready mix x 8.3= 99.6ul
1.4ul Forward Primer x 8.3 = 11.62ul
1.4ul Reverse Primer x 8.3 = 11.62ul
10.2ul H2O x 8.3 = 84.66ul
Aliquot 22ul of master mix into one 8 strip PCR tube
Add 3ul of sample to each tube, the last tube adding H20 as a control
Repeat master mix and strip tube for the second primer set
Samples run on a 1.5% gel once finished for about 1.5 hours
Looks like there is something being amplified, and it does not look like there is contamination, which we had seen before.
After doing these PCRs my samples from GSO beach were almost out, so I want to filter more water and try it on them.