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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

REFERENCE NO: AHC/1998/3/3.2/3.1


TITLE: MYCOPLASMA DECTECTION BY NESTED DOUBLE STEP PCR - INRC -


INTRODUCTION

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 and controls preparation

Materials and solutions

  • 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: (100l total volume per sample): 10l PCR buffer 10X (100mM Tris-HCl, pH 8.3; 500mM KCl, 0.01% gelatin); 10l Mgcl2 25mM; 4.5 l 10%NP40 in sterile distilled H2O; 4.5l 10% TWEEN 20 in sterile distilled H2O; 0.6l proteinase K from a solution 10mg/ml in sterile distilled H2O; 70.4l sterile distilled H2O
  • PBS phosphate buffered saline

Procedure

  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 100l extraction buffer
  4. Incubate the sample at 60C for 1 hour, then 10 min. at 95C
  5. Keep the sample at -20C

 

DNA amplification

Materials and solutions

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

Procedure

  1. Place 20l of sample or controls in 0.5 ml Eppendorf microtubes and add 29 l "PCR mixture first amplification". Prepare the internal control by substituting the sample with sterile distilled H2O
  2. Add 1l Tad DNA polymerase
  3. Add 50 l mineral oil
  4. Amplify the sample in thermal cycler following the described programme:
  5. 1 cycle 90C for 10 min.

    30 cycles 94C for 30 sec.

    60C for 30 sec

    72C for 1 min.

    1 cycle 72C for 10 min.

  6. Analyse 10 l of the amplification product by electrophoresis on agarose gel 2%
  7. Place 10 l of the product of the first amplification in Eppendorf microtube and add 39 l of "PCR mixture second amplification"
  8. Add 1 l Taq DNA polymerase
  9. Add 50 l mineral oil
  10. Amplify the sample in thermal cycler following the described programme:
  11. 1 cycle 90C for 10 min.

    30 cycles 94C for 30 sec.

    60C for 30 sec

    72C for 1 min.

    1 cycle 72C for 10 min.

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

 

PREPARATION OF THE AGAROSE GEL AND ELECTROPHORESIS

Materials and solutions

  • 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 g/l)
  • Loading buffer (0.05% bromophenol blue; 40% sucrose, 0.1 M EDTA pH 8, 0.5% SDS)
  • Polaroid film

Procedure

  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 50C and add 2.5 l 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 l loading buffer to 10 l of the amplification product. Prepare the standard by adding 3 l loading buffer and 8 l sterile distilled H2O to 2 l 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

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