TITLE: Mycoplasma Testing Protocol
AUTHORS: Carolina Lopez

[DESCRIPTION]
Mycoplasma Testing Protocol (Adapted following manufacturer's instructions)

[STEPS]
SECTION: Mycoplasma Testing Protocol
1. Reagents for luminescence test: 
MycoAlert PLUS Detection Kit,  Lonza, cat #LT07-710. 
Positive Control – supernatant from positive sample, 75ul aliquots stored at -80°C
Negative Control – Water or sample from myco-free cell line, NOT unused media
Opaque 96 well plate – Falcon cat #353296
Antibiotic-free media (see materials)

Reagent preparation and storage conditions for MycoAlert Kit Reagent/Substrate: 
Resuspend in provided buffer, aliquot, and store at -80°C.
Room temp – must be used within 5 hours
2-8°C – store for up to 5 days 
-80°C – store for up to 6 months, allow to thaw to room temp without the use of artificial heat, once
thawed - aliquots should not be refrozen 
SECTION: Mycoplasma Testing Protocol
2. Sample Preparation:

Plate cells in a 6-well plate in 2mL of Antibiotic-free media such that cells are 80-100% confluent at 48hr without being over confluent.
At 48hr, collect 1.5 mL of media in 1.5mL tube
Centrifuge at 200xg for 5 minutes
Transfer 1mL of supernatant to a fresh tube
Store at 4°C until testing (within 1 week)
SECTION: Mycoplasma Testing Protocol
3. Sample Testing: 

Allow all reagents to warm to room temperature (~15 minutes)
Load 50µl of sample to a white opaque 96-well plate
 

3. Add 50µl of MycoAlert Plus Reagent to sample and mix
4. Wait 5 minutes
5. Measure luminescence with a 1s integration time, auto gain (Read A)
6. Add 50µl of MycoAlert Plus Substrate to sample and mix
7. Wait 10 minutes
8. Measure luminescence with a 1s integration time, auto gain (Read B)
SECTION: Mycoplasma Testing Protocol
4. Results:

Divide Read B by Read A to produce a ratio 

Ratio / Interpretation
< 1.2 / Negative for Mycoplasma - Okay to keep using
1.2-1.8 / Borderline, residual contamination - Test using second method (PCR)
> 1.8 / Positive for Mycoplasma - Discard cells
 

SECTION: 2nd Testing Method (PCR)
5. Reagents for PCR test: 
Mycoplasma Detection Kit, Southern Biotech, Cat. No 13100-01
Platinum Taq DNA Polymerase, Invitrogen, Cat. No 10966018

Reagent Preparation
Centrifuge all kit component tubes to ensure that material has completely settled to bottom
Reconstitute primer/dNTP mix, positive, and internal control using DNase free water as follows - 
     - Primer/dNTP mix - 260 µL
     - Positive control - 200 µL
     - Internal control - 200 µL
Vortex for 5 seconds to ensure thorough mixing and equilibrate at room temperature for 5 minutes
Vortex, then centrifuge again
Reconstituted reagents should be stored at or below -20ºC; avoid multiple freeze/thaw cycles
SECTION: 2nd Testing Method (PCR)
6. Sample Preparation
Use the sample collected from the 6-well plate in the above step. 
Transfer 100 µL of the cell culture supernatant to a sterile tube; ensure the lid has been tightly sealed to prevent evaporation
Heat the sample at 95ºC for 5 minutes
Quickly spin the sample supernatant at maximum speed for 5 seconds to remove cell debris; the supernatant is now ready to add to the PCR master mix
SECTION: 2nd Testing Method (PCR)
7. Sample Testing
Prepare PCR Master Mix. 
Total volume per reaction is 50 µL (25 µL for 1/2) 
 

When preparing reactions, calculations should include enough reagents for positive and negative controls
Contents of the master mix are provided below -
 

2. Add 48 µL (24 µL for 1/2) of master mix to each tube
3. Add 2 µL (1 µL for 1/2) of DNase-free water as a negative control into the appropriate PCR reaction tube
3. Add 2 µL (1 µL for 1/2) of heat-treated test sample into the appropriate PCR reaction tube
4. Add 2 µl (1 µL for 1/2) of the reconstituted positive control DNA supplied in the kit into the appropriate PCR reaction tube
5. Run according to the thermal cycling program
Incubation times are dependent on the type of polymerase used; the template described below has been optimized for Hot Start Taq polymerase
 
6. Prepare a 1.2% agarose gel containing 0.5µg/mL Ethidium bromide
7. Load 18 µL of each PCR reaction mixed with 3 µL of 6X loading buffer into each well
8. Run at 110 volts for ~30 minutes
9. Detect PCR product bands using ChemiDoc


SECTION: 2nd Testing Method (PCR)
8. Results (Gel Evaluation)
Samples containing Mycoplasma infection will contain a band (or multiple bands) between ~448 bp to ~611 bp
Samples containing the supplied internal control DNA will contain a distinct 270bp PCR product that is clearly distinguishable from the ~488 bp to ~611 bp band(s) found in the positive samples
     - The internal control indicates a successfully performed PCR reaction
     - PCR inhibition may have occurred if the internal control band disappears in some samples, but the band is present in the PCR reaction of the negative control (water added instead of the test sample). If the PCR of a sample is inhibited, the inhibitors can be easily removed by performing a DNA extraction.
If the cell culture is heavily contaminated with Mycoplasma, amplification of the ~488 bp to ~611 bp product(s) may diminish or completely eliminate the 270-bp internal control product
Low-intensity bands smaller than 100 bp indicate the presence of non-specific, self-annealing primers; this does not indicate a positive result and will not affect the precision of the test