Research and Development on Lentivectors for:

• Gene Therapy
• Cell Therapy
• Cell Engineering

Mycoplasma Assay

This method is used to detect mycoplasma in DNA or culture supernatant from cells.

It is cheap, sensitive, reliable and the primers used can detect most strains of mycoplasma.

 

To extract DNA, you can use 1 to 2 millions cells, extracted with a Qiagen DNAeasy kit. Use 1 µl of sample from a 100 µl extract.

If you want to use supernatant, harvest 100 µl of cell culture medium that was in contact with cells for at least 2 consecutive days (3 is best), incubate at 95°C for 5 min and store at -20°C. Note that you cannot use L32 primers as internal control when you use culture supernatants.

L32 primers are from Homo sapiens ribosomal protein L32 pseudogene (Genbank NG_021756)

The PCR mix is as follows:

Red Taq mix 2x                   7.5 µl

Primer mix 10µM                 0.3 µl

H2O                                       6.2 µl

DNA                                         1 µl

Where the primer mix is either MYCO or L32 (internal control).

Primers MYCO

Fwd 5’- GGCGAATGGGTGAGTAACACG

Rev 5’- CGGATAACGCTTGCGACCTAT

Amplicon approx. 500 bp

Primers L-32

Fwd 5’- GTGAAGCCCAAGATCGTCAA

Rev 5’- TTGGTGACTCTGATGGCCAG

Amplicon approx. 400 bp

The PCR program is as follows:

Step 1 - 95°C – 5 min

Step 2 – 95°C – 20 sec

Step 3 – 55°C – 1 min

Step 4 – 72°C – 1 min – from 2 to 4, 32 cycles

Step 5 – 72°C – 10 min

Step 6 – 4°C pause