PCR and junk?

This week I conducted a Polymerase Chain Reaction (PCR) on a sample of the Palo Verde DNA I extracted last week. PCR is a technique used to amplify a sample of DNA. Essentially, PCR creates an extraordinarily large amount of copies of a specific sequence of DNA. I used Universal Rice Primers (URP's) to select specific Simple Sequence Repeats (SSR's) present in the DNA of many species (hence universal rice primer). The primer recognizes these SSR's and singles these sequences out for exponential reproduction. The sample is then introduced into a "Mastermix" that contains free nucleotide bases, and Taq Polymerase, an enzyme that synthesizes the new copies of DNA. The sample, URP's and the mastermix are then placed in a thermal cycler. The thermal cycler runs a program that very specifically cycles the temperature between high and low for certain time frames as follows:

1.     Initial denaturation: 94°C for 3 minutes

2.     Denaturation: 94°C for 1 minute

3.     Annealing: 60°C for 45 seconds

4.     Extension: 72°C for 1 minute

Steps 2-4 repeat 34 times

5.     Final extension: 72°C for 12 minutes

Hold: 4°C indefinitely

The thermal cycler

The Temperature variance is used to assist the reaction's efficiency. The initial high Temperature allows denaturation to occur (separation of the strand of DNA). The cooler Temperature during the annealing stage allows the URP's to bond to the SSR's. During extension, the Taq polymerase adds the new bases that correspond with the nucleotide bases in the DNA strand creating a new segment of DNA. These steps are repeated 34 times so exponential growth occurs in the amount of DNA segments present.

After the PCR was completed, I ran another gel electrophoresis in hopes of seeing good DNA bands. Unfortunately this wasn't the case. The DNA did not appear to travel through the gel. I could see traces of the loading dye that had travelled, so I know that there was not a voltage problem. It is possible the DNA was "junk" DNA, I cannot be sure as yet.  Next week I will run another PCR on the same DNA extraction, as well as complete a new extraction and run it through PCR as well. It is my hope that between these two test I will be able to discern what the issue may be. Until then, here's for hoping...
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