Test eDNA PCRs

Feb 25, 2019

Testing eDNA Extraction PCR Amplification with MiFISH Primers

Test 1 02-20-19

Testing MiFISH Universal and Elasmobranch primers on extracted DNA using “clean” and Test 3 and Test 4 samples using the MIFISH PCR program under CP in our thermocycler (Note that this is not the program specified in the paper)

Try volumes recommended by paper

Sample Tube # Primer
clean 1 U
no DNA 2 U
test 3 3 U
test 4 4 U
clean 5 E
no DNA 6 E
test 3 7 E
test 4 8 E

To each tube:

PCR cycle about an hour

Ran on a 1.5% gel with 100bp ladder

gel

Does not work, also see that we get primer dimer.

Test 2 02-21-19

Trying less primer (1µl) and using the thermocycler program used in the paper. Programed under user MES, program MIFISH UE. Added extra 15 seconds before cycling begins at 98 degrees C for hot start

Sample Tube # Primer
clean 9 U
control 10 U
test 3 11 U
test 4 12 U
clean 13 E
control 14 E
test 3 15 E
test 4 16 E

To each tube:

PCR cycle about an hour

Ran on a 1.5% gel with 100bp ladder

gel

Does not work, also still getting primer dimer. Also, could not have enough DNA in the 1µl added

Test 3 02-25-19

Try two PCRs this time. Make new primer stocks, 10µM, and vacufuge samples to increase their concentration. Trying PCR volumes and program from the paper and PCR volumes and program from Diana already in the PCR machine and using MyTaq mix

Paper PCR

Sample Tube # Primer
clean 17 U
control 18 U
test 3 19 U
test 4 20 U
clean 21 E
control 22 E
test 3 23 E
test 4 24 E

Volumes are the same as the previous PCR volumes

Diana’s Protocol PCR

Sample Tube # Primer
clean 25 U
control 26 U
test 3 27 U
test 4 28 U
clean 29 E
control 30 E
test 3 31 E
test 4 32 E

To each tube:

gel

MiFish, PCR