B: Polymerase Chain Reaction (PCR)

1. Reagents

1. Advantage 2 Polymerase Mix, containing Taq polymerase and PCR buffer (Clontech)
2. dNTP, 25 µM (GenHunter)
3. Forward primer, 10 µM
4. Reverse primer, 10 µM
5. dCTP a-33P (or a-32P) (10 mCi/ml)
6. DEPC-dH₂0 (Research Genetics)
7. High density TBE sample buffer, 5X (Novex)

2. Method

TIP: As with any PCR reaction, it is good practice to run duplicate or triplicate samples to ensure the validity of the PCR result.
TIP: Components and cycling will depend on individual template and primers.

1. Aliquot 3 µl of each cDNA sample into a sterile PCR tube.
2. Prepare sufficient volume of PCR master reaction mixture for all reaction tubes and add 7 µl to each tube.

   PCR master reaction mixture/tube 1.0 µl PCR Buffer 0.8 µl dNTP 0.2 µl Forward primer 0.2 µl Reverse primer 0.2 µl dCTP a-33P (or a-32P) see TIP below 0.2 µl Taq DNA polymerase, 5 U/µl 4.4 µl DEPC-dH20   Total volume = 7 µl

   TIP: The PCR protocol presented here includes incorporation of radioactivity into the PCR products. Radioactivity is necessary for visualization on a denaturing acrylamide gel for low abundant transcripts or when PCR product patterns are complicated (i.e., polymorphic markers for LOH). The amount of radioactivity used in the above protocol often results in visible products in less than 2 hours exposure. For abundant mRNAs, it may be possible to discern products on an ethidium bromide treated agarose gel (replace ³²P or ³³P volume with water).

    

3. PCR Cycling Conditions:

   Cycles Temp. (°C ) Time 1 95 2 min 35 95 X* 72 15 seconds 45 seconds 5 min 1 72 10

   X* is annealing temperature, dependent on the primer used.

4. Store the PCR products at 4°C or continue to step 5.
5. Pour a 6% polyacrylamide sequencing gel while the PCR is cycling.
6. After cycling is complete, add 2.5 µl sample buffer (5X) to samples
7. Denature samples at 95°C for 3 minutes and place directly on ice.
8. Load 3.5 µl sample on gel and run at 1600 V to desired distance.
9. Dry gel and expose to phosphoroimager screen or film as described under LOH protocol.

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