( Use 20 l of the supernatant...)Preparation of PCR Quality Template DNA from a Wide Range of Tissues Using InstaGene Matrix

Notes: Store remainder at -20 C for subsequent PCRs. Repeat step 6 when reusing the preparation (allow to thaw first).

PCR
Primers used in this experiment were designed to amplify a 120 base pair fragment of the Canine Pancreatic Lipase Gene (Mickel et al., 1989). Amplification was carried out using 2 Units AmpliTaq DNA polymerase (Perkin-Elmer) mixed with an equal volume of TaqStart antibody (Clontech) per reaction in a total reaction volume of 50 l, containing 50 pmol each primer and 200 mol/L of each deoxynucleoside triphosphate in 10 mM Tris (pH 8.4), 50 mM KCl, and 1.5 mM MgCl₂. Thermal cycling was performed on a Perkin-Elmer 9600. Initial denaturation was at 94 C for 5 minutes, followed by 40 cycles of 94 C for 30 seconds, 57.5 C for 1 minute, then a final extension of 72 C for 7 minutes.

Results

All tissues examined produced a clear band of the correct size. The variation in the brightness of the band does not appear to correspond to the amount of DNA template present in each reaction. Apart from the positive control (A72 cell line), there are two template copies per cell for each tissue. The most likely cause of the variation in intensity of the band obtained is therefore inhibitory factors present within the sample. It is important not to exceed the capacity of the InstaGene matrix by using too much tissue in the initial preparation. Exceeding the matrix capacity may cause PCR-inhibitory factors normally removed by the procedure to remain in the supernatant. Carryover of these inhibitory factors may affect amplification in the subsequent PCR reaction.
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