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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

• 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: (100µl total volume per sample): 10 µl PCR. Buffer 10X (100mM Tris-HCl, pH 8.3; 00mM KCl; 0.01% gelatin); 10µl MgCl₂ 25mM; 4.5 µl 10% NP40 in sterile distilled H2O; 4.5 µl 10% TWEEN 20 in sterile distilled H₂O; 0.6 µl proteinase K from a solution 10mg/ml in sterile distilled H₂O; 70.4 µl sterile in distilled H₂O
• PBS phosphate buffered saline

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 µl extraction buffer
6. Incubate the sample at 60°C for 1 hour, then 10 min. at 95°C
7. Keep the sample at -20°C

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

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

 

4. Amplify the sample in thermal cycle following the described programme:

1 cycle 90°C for 10 min.

30 cycles 96°C for 30 sec.

63 °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%

• 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