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This assay is based on the use of a pool of primers (9 outers + 9 inners) that amplify respectively a fragment of 504-519 bp (outer primers) and 318-333 bp (inner primers) of sequences coding for a conserved region of the 16S rRNA of at least 25 different species of Mycoplasma

• Sample: supernatant with some cells of adherent or suspension cell line cultured for at least 3 passages in the absence of antibiotics
• Positive control: supernatant with some cells of a contaminated cell line
• Negative control: supernatant with some cells of a non contaminated cell line
• Extraction buffer: (100µl total volume per sample): 10µl PCR buffer 10X (100mM Tris-HCl, pH 8.3; 500mM KCl, 0.01% gelatin); 10µl Mgcl2 25mM; 4.5 µl 10%NP40 in sterile distilled H₂O; 4.5µl 10% TWEEN 20 in sterile distilled H₂O; 0.6µl proteinase K from a solution 10mg/ml in sterile distilled H2O; 70.4µl sterile distilled H₂O
• PBS phosphate buffered saline

1. Place 1 ml of the cell culture supernatant in a sterile Eppendorf tube
2. Centrifuge at 13000 rpm for 6 min.
3. Resuspend pellet in 100µl extraction buffer
4. Incubate the sample at 60°C for 1 hour, then 10 min. at 95°C
5. Keep the sample at -20°C

• PCR mixture first amplification (29µl volume per sample): 1µl of each primer (50 pmol/µl), total volume 9µl; 5µl dNTP 2mM; 5 µl PCR Buffer 10X (100 mM Tris-HCl, pH8.3; 500mM KCl; 0.01% gelatin); 3µl MgCl2 25 mM; 7 µl sterile distilled H2O
• PCR mixture second amplification (39µl volume per sample): 1µl of each primer (50 pmol/µl), total volume 9µl; 5µl dNTP 2mM; 5 µl PCR Buffer 10X (100 mM Tris-HCl, pH8.3; 500mM KCl; 0.01% gelatin); 3µl MgCl2 25 mM; 17 µl sterile distilled H2O
• Taq DNA polymerase in PCR buffer 1X, 1 µl (1.25 U/ml)
• Samples and controls 20µl (first amplification) or 10µl (second amplification)
• Mineral oil 50 µl

1. Place 20µl of sample or controls in 0.5 ml Eppendorf microtubes and add 29 µl "PCR mixture first amplification". Prepare the internal control by substituting the sample with sterile distilled H₂O
2. Add 1µl Tad DNA polymerase
3. Add 50 µl mineral oil
4. Amplify the sample in thermal cycler following the described programme:

1 cycle 90°C for 10 min.

30 cycles 94°C for 30 sec.

60°C for 30 sec

72°C for 1 min.

1 cycle 72°C for 10 min.

5. Analyse 10 µl of the amplification product by electrophoresis on agarose gel 2%
6. Place 10 µl of the product of the first amplification in Eppendorf microtube and add 39 µl of "PCR mixture second amplification"
7. Add 1 µl Taq DNA polymerase
8. Add 50 µl mineral oil
9. Amplify the sample in thermal cycler following the described programme:

1 cycle 90°C for 10 min.

30 cycles 94°C for 30 sec.

60°C for 30 sec

72°C for 1 min.

1 cycle 72°C for 10 min.

10. Analyse 10 µl of the amplification product by electrophoresis on agarose gel 2%

• Agarose
• TBE 10X (108g Tris base, 50g boric acid, 40 ml EDTA 0,5 M pH8 in 1 liter distilled H₂O)
• Ethidium bromide 10mg/ml
• Molecular weight marker: phiX 174 RF DNA digested with Hae III (0.5 µg/µl)
• Loading buffer (0.05% bromophenol blue; 40% sucrose, 0.1 M EDTA pH 8, 0.5% SDS)
• Polaroid film

1. Dissolve 1 g agarose in 50 ml TBE 1X (agarose 2%) by boiling 3 - 5 min.
2. Cool the solution to a temperature of about 50°C and add 2.5 µl ethidium bromide
3. Pour onto an electrophoresis plate with the well comb in place and allow about 20 min. for the gel to set. Remove the comb
4. Transfer the solid gel to the electrophoresis chamber, add enough TBE 1X to cover the gel
5. Add 3 µl loading buffer to 10 µl of the amplification product. Prepare the standard by adding 3 µl loading buffer and 8 µl sterile distilled H₂O to 2 µl molecular weight marker phiX174/Hae III
6. Load the samples into the wells
7. Run the gel on constant voltage at 120 V for 10 min. and 100 V for about one hour.
8. Photograph the gel on the UV illuminator using a Polaroid camera

Guidelines prepared for CABRI by CERDIC, DSMZ, ECACC, INRC, November 1998
Page layout by CERDIC
Copyright CABRI, 1998