Polony PCR Amplification

This section outlines the protocol for single-molecule amplification within the acrylamide gel.  The protocol given here may differ in specifics from what is presented here, but reflects what we are doing currently (Summer 2003).  We have tried to include excessive detail here but let us know if anything is unclear.  The general steps are:

1.    Cast acrylamide gels
2.    Diffuse in PCR reagents
3.    PCR amplification
4.    Slide clean-up


Further Protocol 

Long PCR Protocol Reagents and Guidelines

General Guidelines for Long PCR Conditions and Enzyme Mixtures
Efficient Long PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Following the results of Cheng et al. (1) we have had success using Tth (ABI/Perkin-Elmer) as the main-component polymerase and Vent (New England Biolabs) as the fractional-component polymerase. Other combinations of proofreading and non-proofreading polymerases have been used successfully for many applications. The buffer listed below works well with Tth and Vent, but not with others. If you are interested in using other polymerases make sure that you use compatible buffers. Error rates for other polymerases can be found at http://research.nwfsc.noaa.gov/protocols/taq-errors.html 

Read Full Protocol Here

TEM-PCR Method,Technology and Testing

What is TEM-PCR Technology?

The TEM-PCR stands for Target Enriched Multiplexing Polymerase Chain Reaction. TEM-PCR is a molecular multiplex PCR technology used Molecular Differential Detection. By TEM-PCR the molecular targets can be amplified in one reaction. This technology is advantageous over the RT-PCR Technology because multiple molecular targets can be amplified specifically in a single reaction. This makes it a important tool for detection of molecular markers of pathogens.

In last few years improvements have been done in this technology. Benson et. al. compared the ResPlex I assay kit of Qiagen with RT-PCR in the detection of various bacterial strains of respiratory disease and found that it is less sensitive than real-time PCR but has advantage over multiple assays [ref]. Deng et. al. detected more S. pneumoniae (32 vs. 7) and H. influenzae (29 vs. 23) by TEM-PCR than did culture [ref]. Related: Overlap Extension PCR