27 February 2002
- Forward Gene Specific Primer (IDT)
- Reverse Gene Specific Primer (IDT)
- Random decamer primer (from Ambion RetroScript Kit: 1710, $206.00/kit or Ambion, 5722G, $50.00/80Âµl)
- 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)
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)
Prepare of Primer Mixes (for desired gene) per reaction:
Forward Primer 2 Âµl
Reverse Primer 2 Âµl
Total Primer Mix 4 Âµl
Prepare of 10s rRNA PCR Primer:Competimer Mix (e.g., 1:9 Ratio):
18 S rRNA PCR Primer 1Âµl
Competimer 9 Âµl
Total 1:9 Mix 10 Âµl
Prepare PCR Reaction Cocktail (ideal to include 4 controls):
10x Complete PCR Buffer 5 Âµl
2.5 mM dNTP 4 Âµl
Taq Polymerase 0.2 Âµl
18 S Primer:Competimer Mix 4 Âµl
RNase-free H2O 4 Âµl
Total 45 Âµl
Mix by pipetting up and down, centrifuge briefly
For your Controls:
Aliquot 45 Âµl into 4 PCR (0.2 ml) tubes marked C1, C2, C3, C4 (or aliquot 180 Âµl (45 Âµl x 4) into a tube with 16ul 18 S primer pair and aliquot 49 Âµl into 4 tubes)
Add 4 Âµl 18 S Primer pair to each tube then add 1 Âµl of the
C1 = 1 Âµl of (+) RT Control cDNA
C2 = 1 Âµl of (-) RT Mock Control cDNA
C3 = 1 Âµl of (-) RT LAU Control cDNA
C4 = 1 Âµl of (-) PCR Control (ddH2O)
Total reaction mix/tube per tube = 50 Âµl
Add 4 Âµl of gene specific primer mix per cocktail mix. Mix well by pipetting up and down.
Aliquot 49 Âµl into a PCR tubes
Add 1 Âµl of appropriate cDNA. and mix by pipetting up and down, centrifuge briefly and keep on ice.
Close tube lid tightly and put into PCR machine at 95Â°C.
Run the tubes at the pre-determined optimal cycles per gene.
Perform PCR under the following conditions:
- 95Â°C for 3 minutes
- 95Â°C for 45 seconds
- 58Â°C for 45 seconds (check annealing temp for gene)
- 72Â°C for 45 seconds
REPEAT for OPTIMAL CYCLES (pre-determined)
- 72Â°C for 10 minutes
- 4Â°C Forever
Remove tubes at the appropriately designated cycle and kept on ice or at 4Â°C until gel can be run.
Analyze 10 Âµl PCR Product by mixing with 2.5 Âµl 5x dye in a 2% agarose gel with 3.5 Âµl 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 Âµl 1x TAE and 25 Âµl Sybr Green) for 1.5 hours while shaking.
- Observe gel under UV light and photograph the image. Also, scan the gel under STORM Fluorimager to be able to analyze quantitative differences.