News
Counting PCR: A new method to obtain absolute DNA copy number without a standard curve
July 21, 2015
John SantaLucia, Jr. and Gregory J. Boggy DNA Software, Inc., Ann Arbor MI 48104 Abstract DNA Software has discovered how to analyze the shape of a PCR curve to reveal the absolute copy number of DNA at cycle zero. This... Read more ›
Quick Guide to the Precision and Accuracy of results from qPCR CopyCount
July 21, 2015
Purpose: This Quick Guide to the Output provides you with the basic description of QRT PCR data analysis and how to interpret the output results from qPCR CopyCount. Summary: An understanding of the difference between precision and accuracy is critical to... Read more ›
Two-step Assay Calibration Procedure for TaqMan Assays
July 21, 2015
Introduction The following procedure is performed on each new qPCR assay that will be analyzed by qPCR CopyCount. The method is called “2-step” because it involves two qPCR reactions: one preliminary PCR with 4 replicates to get a rough concentration,... Read more ›
Quick Start Guide for qPCR CopyCount
July 21, 2015
Purpose: This CopyCount Quick Start Guide provides the basic information and best practices for running qPCR CopyCount. Best practices for setting up your qPCR plate We recommend that each sample be run with at least 4 replicates. This allows for outliers... Read more ›
Multiplex PCR Optimization
July 21, 2015
Myth 7: Multiplex PCR Can Succeed by Optimization of Individual PCRs Not too many people believe this myth, and yet their actions are somewhat irrational as they proceed to immediately use that approach to try to experimentally optimize a... Read more ›
Amplification Efficiency for primers Is Not Exponential
July 21, 2015
Myth 6: At the End of PCR, Amplification Efficiency Is Not Exponential Because the Primers or NTPs Are Exhausted or the Polymerase Looses Activity PCR amplification occurs with a characteristic “S” shape. During the early cycles of PCR, the amplification... Read more ›
Why BLAST Search is a Myth for Determining the Specificity of a Primer
July 21, 2015
Myth 5: A BLAST Search Is the Best Method for Determining the Specificity of a Primer To minimize mispriming, several PCR texts suggest performing a BLAST search, and such capability is a part of some primer design packages such... Read more ›
Primer Dimer Artifacts Are Due to Dimerization
July 21, 2015
Myth 4: “Primer Dimer” Artifacts Are Due to Dimerization of Primers A common artifact in PCR is the amplification of “primer dimers.” The most common conception of the origin of primer dimers is that two primers hybridize at their... Read more ›
Designing Forward and Reverse Primers to Have Matching Tm
July 21, 2015
Myth 3: Designing Forward and Reverse Primers to Have Matching Tm’s Is the Best Strategy to Optimize for PCR Nearly all “experts” in PCR design would claim to believe in myth 3. Most current software packages base their design strategy... Read more ›
Best Methods for Predicting Hybridization Tm
July 21, 2015
Myth 2: Best Methods for Predicting Hybridization Tm Best methods for predicting hybridization Tm are essentially equivalent in accuracy. The melting temperature, Tm, of duplex formation is usually defined as the temperature at which half the available strands are in... Read more ›