Testing for mycoplasma in cell culture by PCR

Nuclease free water

Go-Taq Green master mix x2 (Promega M712B)

Fwd oligo:  Combine to a final concentration of 10 uM.

Rev oligo: Combine to a final concentration of 10 uM.

Negative control (e.g. medium only)

Positive control (medium from infected culture)

Agarose powder

Running buffer (recipe)

Pipetors 20μl, 10μl

Filter tips

Eppendorf tubes

PCR tubes 0.2ml

Heating block (95°C)

PCR machine

Mini-centrifuge

Microspin benchtop centrifuge

Gel electrophoresis aparatus

UV imaging (3rd floor)

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):

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.