Controls Used To Monitor

Controls are used to monitor the effectiveness of the chosen experimental conditions and/or the technique of the experimenter. These controls typically

Table 4.2

Typical components for PCR amplification.

Reagent

Tris-HCl, pH 8.3 (25°C) Magnesium chloride Potassium chloride

Deoxynucleotide triphosphates (dNTPs) DNA polymerase, thermal stablea Bovine serum albumin (BSA) Primers Template DNA

Optimal Concentration

200 |M each dATP, dTTP, dCTP, dGTP 0.5-5 U 100 |g/mL 0.1-1.0 |M

1-10 ng genomic DNA

aTaq and Taq Gold are the two most common thermal stable polymerase used for PCR.

include a 'negative control', which is the entire PCR reaction mixture without any DNA template. The negative control usually contains water or buffer of the same volume as the DNA template, and is useful to assess whether or not any of the PCR components have been contaminated by DNA (e.g., someone else in your lab). An extraction 'blank' is also useful to verify that the reagents used for DNA extraction are clean of any extraneous DNA templates (Presley and Budowle 1994).

A 'positive control' is a valuable indicator of whether or not any of the PCR components have failed or were not added during the reaction setup phase of experiments conducted. A standard DNA template of known sequence with good quality DNA should be used for the positive control. This DNA template should be amplified with the same PCR primers as used on the rest of the samples in the batch that is being amplified. The purpose of a positive control is to ensure confidence that the reaction components and thermal cycling parameters are working for amplifying a specific region of DNA.

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