27 February 2002

 

Reagents:

  • Forward Gene Specific Primer (IDT)
  • Reverse Gene Specific Primer (IDT)
  • Random decamer primer (from Ambion RetroScript Kit: 1710, $206.00/kit or Ambion, 5722G, $50.00/80µl)
  • RNase Inhibitor (from Ambion RetroScript Kit: 1710, $206.00/kit)
  • Reverse Transcriptase (M-MLV) (5x RT Buffer from Ambion RetroScript Kit: 1710, $206.00/kit)
  • 10x PCR Reaction Buffer (from Ambion RetroScript Kit: 1710, $206.00/kit)
  • 50 mM MgCl2 (from Ambion RetroScript Kit: 1710, $206.00/kit)
  • SuperTAQ DNA Polymerase (Ambion, 2050, $48.00/50 U or 2052, $190.00/250U)
  • 18S rRNA PCR Primer Pair (Ambion kit, 1716 $155.00/kit)
  • 18S PCR Competimers (Ambion kit, 1716 $155.00/kit)
  • Sybr Green Stain (FMC Bioproduct, 50513, $146.00/box)

Protocol:

Using the cDNA from the previous RT PCR, optimal cycles were determined for gene of interest. The optimal 18S rRNA PCR Primer:Competimer ratio will need to be optimized per gene primer pair (start with 1:9, 2:8, 3:7 and reduce ratio again if rare primer)

  1. Prepare of Primer Mixes (for desired gene) per reaction:

    Forward Primer        2 µl
    Reverse Primer         2 µl
    Total Primer Mix       4 µl

  2. Prepare of 10s rRNA PCR Primer:Competimer Mix (e.g., 1:9 Ratio):

    18 S rRNA PCR Primer     1µl
    Competimer                       9 µl
    Total 1:9 Mix                    10 µl

  3. Prepare PCR Reaction Cocktail (ideal to include 4 controls):

                                             per reaction:
    10x Complete PCR Buffer            5 µl
    2.5 mM dNTP                              4 µl
    Taq Polymerase                         0.2 µl
    18 S Primer:Competimer Mix        4 µl
    RNase-free H2O                           4 µl
    Total                                            45 µl
    Mix by pipetting up and down, centrifuge briefly

For your Controls:

  1. Aliquot 45 µl into 4 PCR (0.2 ml) tubes marked C1, C2, C3, C4 (or aliquot 180 µl (45 µl x 4) into a tube with 16ul 18 S primer pair and aliquot 49 µl into 4 tubes)

    Add 4 µl 18 S Primer pair to each tube then add 1 µl of the following:
    C1 = 1 µl of (+) RT Control cDNA
    C2 = 1 µl of (-) RT Mock Control cDNA
    C3 = 1 µl of (-) RT LAU Control cDNA
    C4 = 1 µl of (-) PCR Control (ddH2O)
    Total reaction mix/tube per tube = 50 µl

  2. Add 4 µl of gene specific primer mix per cocktail mix. Mix well by pipetting up and down.
  3. Aliquot 49 µl into a PCR tubes
  4. Add 1 µl of appropriate cDNA. and mix by pipetting up and down, centrifuge briefly and keep on ice.
  5. Close tube lid tightly and put into PCR machine at 95°C.
  6. Run the tubes at the pre-determined optimal cycles per gene.
  7. Perform PCR under the following conditions:
    1. 95°C for 3 minutes
      start cycles:
    2. 95°C for 45 seconds
    3. 58°C for 45 seconds (check annealing temp for gene)
    4. 72°C for 45 seconds
      REPEAT for OPTIMAL CYCLES (pre-determined)
    5. 72°C for 10 minutes
    6. 4°C Forever
  1. Remove tubes at the appropriately designated cycle and kept on ice or at 4°C until gel can be run.
  2. Analyze 10 µl PCR Product by mixing with 2.5 µl 5x dye in a 2% agarose gel with 3.5 µl Ethinium Bromide in 1x TAE buffer.
    • Run for 1.5 hours (or until ready) at 100V or overnight at 25V.
    • Stained gel with Sybr Green (250 µl 1x TAE and 25 µl Sybr Green) for 1.5 hours while shaking.
  1. Observe gel under UV light and photograph the image. Also, scan the gel under STORM Fluorimager to be able to analyze quantitative differences.
Department of Microbiology
School of Medicine
University of Washington
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