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LABORATORY PROCEDURES FOR ANIMAL & HUMAN CELL LINES

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


TITLE: SPECIES CONFIRMATION FOR HUMAN CELL LINES BY HLA DQ ALPHA AMPLIFICATION


INTRODUCTION

This assay is based on the use of a pair of primers that amplify a fragment of 229 bp of sequences coding for human DQ alpha molecules

 
Sample and controls preparation

Materials and solutions

  • Sample: suspension of 5 x 10 5 cells of adherent or suspension cell lines, 1 ml
  • Positive control: suspension of 5 x 10 5 cells of a human cell line, 1 ml
  • Negative control: suspension of 5 x 10 5 cells of a non human cell line, 1 ml
  • Extraction buffer: (100l total volume per sample): 10 l PCR. Buffer 10X (100mM Tris-HCl, pH 8.3; 00mM KCl; 0.01% gelatin); 10l MgCl2 25mM; 4.5 l 10% NP40 in sterile distilled H2O; 4.5 l 10% TWEEN 20 in sterile distilled H2O; 0.6 l proteinase K from a solution 10mg/ml in sterile distilled H2O; 70.4 l sterile in distilled H2O
  • PBS phosphate buffered saline

 

Procedure

  1. Place 1 ml of the sample in a sterile Eppendorf tube
  2. Centrifuge at 13.000 rpm for 6 min.
  3. Discard supernatant and resuspend pellet in 1 ml PBS
  4. Centrifuge at 13.000 rpm for 6 min.
  5. Repeat the operation and resuspend pellet in 100 l extraction buffer
  6. Incubate the sample at 60C for 1 hour, then 10 min. at 95C
  7. Keep the sample at -20C

 

DNA AMPLIFICATION

Materials and solutions

  • PCR mixture (78 l volume per sample): 10 l of each primer (DQA AmpA, DQA AmpB) (25 pmol/l); 10 l PCR buffer 10X (100mM Tris-HCl, pH 8.3; 500mM KCl; 0.01% gelatin), 6 l MgCl2 25 mM, 10 l DMSO, 22 l sterile distilled H2O
  • Taq DNA polymerase in PCR buffer 1X, 2 l (1.25 U/l)
  • Samples and controls 20 l
  • Mineral oil 50 l

Procedure

  1. Place 20 l of sample or controls in 0.5 Eppendorf microtubes and add 78 l "PCR mixture". Prepare the internal control by substituting the sample with sterile distilled H2O
  2. Add 2 l Taq DNA polymerase
  3. Add 50 l mineral oil
  4.  

  5. Amplify the sample in thermal cycle following the described programme:
  6. 1 cycle 90C for 10 min.

    30 cycles 96C for 30 sec.

    63 C for 30 sec.

    72 C for 1 min.

    1 cycle 72 C for 10 min.

  7. 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


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