Materials and Reagents

Nuclease free water
Go-Taq Green master mix x2 (Promega M712B)
Fwd oligo: Combine to a final concentration of 10 uM.

CGCCTGAGTAGTACGTTCGC
CGCCTGAGTAGTACGTACGC
TGCCTGAGTAGTACATTCGC
TGCCTGGGTAGTACATTCGC
CGCCTGGGTAGTACATTCGC
CGCCTGAGTAGTATGCTCGC
Rev oligo: Combine to a final concentration of 10 uM.

GCGGTGTGTACAAGACCCGA
GCGGTGTGTACAAAACCCGA
GCGGTGTGTACAAACCCCGA
Negative control (e.g. medium only)
Positive control (medium from infected culture)
Agarose powder
Running buffer (recipe)

Equipment

Procedure

Collect 100μl of cell culture media from plate into eppendorf tube.
Boil samples to 95°C for 5min
Centrifuge at top speed for 2min.
Prepare PCR mix as follows (multiply by the number of samples):
Reagent volume
Go-Taq Green master mix x2
10μl
Fwd primer 1.5μl
Rev primer 1.5μl
Sample 1μl
Water 6μl
total: 20μl
Don't forget to include negative & positive controls.
Use the program Sandi-->myco1. program details:
95/ 5min, (95/30 secs, 50/30 secs/ 72/35 secs) X 5 cycles, (95/30 secs, 56/20 secs/ 72/30 secs) X 25 cycles, 72/1 min, 4/hold
Once done, load the samples directly on gel and run for 10-15min at 300/250V.
Image gel.

Recipes