Mycoplasma test --- PCR

The following is the ATCC mycoplasma PCR kit protocol.

1. Grow cells in antibiotic-free medium for at least two passages.

2. For adherent cells, grow ~5000 cells in T25 flask for ~48 hours.

3. Scrape cells into the medium (no Trypsin-EDTA). Place 1 ml of culture (~2000 cells) in a microcentrifuge tube.

4. For suspension cells, place ~5000 cells. (Cells can be stored at -80 C for up to 6 months.)

5. Centrifuge cells at 12,000 rpm for 20 minutes in 4 C.

6. Carefully discard supernatant.

7. Resuspend pellet in 100 ml Lysis buffer by vortexing.

8. Heat to 95 C for 10 minutes.

9. Use immediately or store at -80 C.

10. Prepare a 1:100 and 1:1000 dilution of the two control DNA.

11. Setup PCR reactions. Make a master mix first:

1.1X Taq Polymerase Buffer
( including 2.2 mM MgCl2 and 0.22 mM dNTPs)
45 ml
First Stage Primer 1 ml
PCR DNA Polymerase (5 units/ml) 0.2 ml
Total volume 46 ml

12. Mix 5 ml cell/mycoplasma DNA or control DNA (1:100 and 1:1000) with 45 ml of the master mix. Also include a negative control.

13. Run PCR using the following program:

Step 1 Denaturation 94 C 2 minutes
Step 2 Denaturation 94 C 30 seconds
Step 3 Annealing x C 30 seconds
Step 4 Extension 72 C 1 minute
Step 5 Repeat step 2-4 29 times
Step 6 Storage 4 C

14. Reamplify the first round PCR product using the Second Stage Primers. The conditions are the same as the first round except that the first round products are used as templates and the Second Stage Primers replace the First Stage Primers.

15. Run 10 ml of the second round PCR products on an agarose gel. A band of the size 200-500 bp suggests a mycroplasma contamination.

16. Digest the second round PCR products to confirm the mycoplasma species.