PCR Technologies Protocols Table of Contents
During PCR assay preparation, nonspecific amplification can occur due to binding of PCR primers to nonspecific templates and from formation of primer dimers which result from using other primer molecules as templates. The protocols presented in this section both adopt controlled PCR activation to allow users to minimize nonspecific amplification while increasing target yield and specificity. Two alternative methods are presented to control amplification of the specific product, these are enzyme and dNTP mediated Hot Start PCR.
When using hot start Taq DNA polymerase, the enzyme remains inactive until heated. Hot Start DNA polymerase control is achieved by chemical or antibody modification of the enzyme. Chemically modified hot start enzymes require up to 10 minutes activation whereas antibody mediated hot start enzymes are activated within 1 minute.
JumpStart™ Taq DNA Polymerase is an antibody-inactivated, hot start enzyme. During the initial denaturation step of the PCR, the antibody is also denatured and dissociates from the DNA Polymerase, therefore enzyme activity is restored. The resulting PCR exhibits a higher specificity and yield5.
Figure P2-17.Resolution of DNA Size Standards Through Agarose Gel.
*Note: Magnesium chloride is added separately if not already in the PCR buffer or when previous optimization has revealed a requirement for a higher concentration.
b. Combine reaction components into a 1.5 mL microcentrifuge tube on ice.
*Note: Elongation time is dependent upon amplicon size: 30 sec for up to 500 bp. Add 1 min for each additional 1 kb.
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