Reagents


Provided by Mycoplasma Plus PCR Primer Set (Stratagene, #302008, $299):
  • Rehydration buffer, store at RT
  • PCR primers, rehydrate with 200 µl rehydration buffer, store at -20°C
  • Internal control, rehydrate with 100 µl rehydration buffer, store at -20°C
  • Positive control, rehydrate with 100 µl rehydration buffer, store at -20°C
  • StrataClean Resin, store at 4°C
To be supplied by user:
  • 0.6 ml tubes or 1.5 ml screw cap tubes
  • PCR reagents (i.e. dNTP, Biolase Taq, MgCL2,10x reaction buffer)
  • UV-irradiated ddH2O
  • 2% agarose gel (3 g agarose, 3ml 50x TAE, 147 ml H2O, 75 µl (0.5 µg/µl) Ethidium Bromide)
  • loading buffer

   Protocol


A)   Template Preparation

  1. Centrifuge all supernatant samples to pellet any cell debris as this can inhibit the PCR.
  2. Transfer 100 µl cell supernatant to a 0.6 ml tube (screw cap tubes for viral supernatants). Close tube tightly to prevent opening during heating step.
  3. Boil (or heat to 95°C) supernatant for 5 minutes. Spin tube briefly (2-5 seconds) in a microcentrifuge.
  4. Resuspend the StrataClean Resin by vortexing for 30 seconds until no pellet remains. Add 10 µl of resin to the extract. Mix the resin and the extract by gently flicking the tube. Briefly centrifuge for 5-10 seconds to pellet the resin.
  5. Remove the supernatant to a fresh tube and dilute (optional) 1:10 with UV-irradiated water Per PCR, 5 µl of diluted template is required. Avoid aspirating the resin into the aliquot. Store the template at 4°C until use.

 

B)   Mycoplasma PCR

The amount for one reaction for Mycoplasma PCR is 50 µl.  Prepare a premix of reagents for multiple reactions.  The amount for the premix reagents is 45 µl per reaction.  The amount of each reagent for premix is as follows:

 

          Water                  32.1 µl

          10x RB                5.0 µl

          MgCl2 (25 mM)    3.0 µl

          DNTP (20 mM)    0.5 µl

          Primers (25 µM)   2.0 µl

          Internal control     2.0 µl

          Biolase (5 U/µl)    0.4 µl

                                         45 µl/reaction

 

  1. Calculate volume of premix needed for all samples, one positive control, one negative control (water) and one extra reaction.
  2. Thaw PCR reagents from freezer (except Biolase Taq). Make premix in one tube.
  3. Add Biolase Taq into premix tube, mix and aliquote into PCR tubes.
  4. Add 5 µl test sample, positive control or negative control into the tubes.
  5. If not using thermal cycler with heated lid, add 2 drops of mineral oil.
  6. Keep tubes on ice until ready for cycling.
  7. Start PCR cycling. The following PCR program yields optimal amplification of the 874bp product:

Segment

Cycles

Temperature

Duration

1 1 94°C 2 minutes


50°C 2 minutes


72°C 2 minutes
2 40 94°C 1 minute


50°C 1 minutes


72°C 2 minutes

  1. Store samples at 4°C until ready to run agarose gel.
  2. Make 150 ml of 2% agarose gel.
  3. Prepare samples for loading: 2 µl 5x loading buffer, 3 µl H2O, 5 µl PCR products (or 2 µl 1kb, φx174 or HindIII marker).
  4. Load the samples and run the gel for 1 - 1.5 hours at 100 volts.
  5. Visualize banding pattern: positive control = 874bp, internal control = 1000bp.

     

    For specifics on appropriate handling and waste procedures please see the online chemical SOPs or our waste and spill notebook located in room 352.

Department of Microbiology
School of Medicine
University of Washington
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