Many different conditions were tested with the first of the designed primers.  After amplification results were obtained most alterations to the PCR program were limited to adjustment of the annealing temperature.

During all PCR runs described below the thermocycling program was as follows:

1. 45 sec @ 94^o C (denaturing the DNA - breaking the double strands apart)
2. 1 min @ 54-72^o C (annealing of the primers to the template/iguana DNA)
3. 1 min @ 72^o C (extension/replication of the DNA extending from where the primers have bound)
4. 35 more repetitions of steps 1-3
5. 9 min @ 72^o C (final extension period - I have never heard a satisfactorily logical explanation of what this is for or what it is supposed to accomplish . . .)
6. Hold @ 4^o C

This lowest annealing temperature (53^o C) allowed the primers to bind at many different locations within the genome.  For this reason there are MANY bands of different sized DNA fragments in each lane.

This picture also shows the "ladder" (on the left side and in the middle) used to estimate the size of the DNA fragments.  The ladder is a mixture of many different sizes of DNA fragments of known sizes.  The size (number of base pairs) in the ladder fragments is written in on the far left side.

Additionally, this picture shows a comparison that was made between 3 different extraction techniques (standard salting out - not specially marked, boiling - B, and microwaving - M).  For additional information on these techniques see DNA extraction techniques tested.
 

This picture has the same samples as found above run at an annealing temperature of 55^o C.  In the samples of standard (salting out) extraction there are slightly fewer bands.  However, more interestingly the boiled samples produced no bands during the second PCR run (which they had produce during the first PCR run at 53^o C).  This may likely be attributed to the unpurified state of the sample solution.  The DNA was not separated from any active enzymes found within the cells or fluid of the blood sample that might degrade or further damage the DNA.  (This is one explanation anyway.)  Microwaving the blood samples prior to direct PCR still did not appear to work.
 

This gel contained PCR products run at 3 higher annealing temperatures (57, 59, & 61^o C).  The general trend is clear.  As the annealing temperature is increased the number of bands produced decreases.  However, for some reason the number of bands appeared to increase for male 96 at 59^o and for male 290 at the higher annealing temperatures. (???)

An additional point of interest is the location of the xylene cyanol FF dye in the middle of the gel.  This band is visible if the dye is not completely lost from the gel during de-staining.  The bromophenol blue dye may also be apparent if it has not run off the end of the gel.  These dyes are used to visually monitor the relative movement of the DNA through the gel (so you know when to turn off the power and stop the run).  The relative speed of movement for each dye is known for gels of various concentrations.  In higher concentration gels (that are thicker in consistency) the dye and DNA move more slowly and separation of the short pieces of DNA is better accomplished.  Thin/low concentration gels are useful for simply checking to see if DNA is present or for separating long DNA fragments.
 

As the annealing temperature is raised the number of bands continues to decrease. . . .  Oh, if you are wondering about what those circles and lines in the background are, they are artifacts due to the plastic that I wrap around the gel to hold it onto one of the glass plates that it was run on.  I have found that this makes it easier to transport and manipulate the gel during the picture taking.  It is always easy to pick up the gel after taking the picture and there is never a mess to clean up on the apparatus.  However, it is important to note that the gel itself must be against the glass (with only the plastic between it and the UV light), since the UV does not penetrate the glass plate and reach the gel if the glass is on the bottom.  But once the UV reaches the gel and hits the ethidium bromide the fluorescence produced may be seen through the glass.
 

This is just a closer look at the lanes that had samples run at the 64^o C annealing temperature.  Again, the 1 kb ladder fragment lengths (in base pairs - bp) are identified along the far left side.
 

Once again, the number of bands produced is seen to decrease as annealing temperature increases. . . .
 

And finally, as the annealing temperature approaches the extension temperature (72^o C) the bands produced completely disappear.  So a 68^o C annealing temperature appears to be optimal at this point.  (The bands of interest - just below the 298 bp marker - are the only ones remaining, or nearly.)  :)
 

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