[...]... Kolmodin, L., Cheng, S., and Akers, J (1995) GeneAmp XL PCR Kit Amplifications: A Forum for PCR Users (The Perkin-Elmer Corporation) 13, 1–5 PCR Primer Design 19 2 Computer Programs for PCR Primer Design and Analysis Bing-Yuan Chen, Harry W Janes, and Steve Chen 1 Introduction 1.1 Core Parameters in Primer Design 1.1.1 Tm, Primer Length, and GC Content (GC %) Heat will separate or “melt” double-stranded... name a few The reverse dot-blot method combines PCR amplification with nonradioactive detection (35) The introduction of fluorescent dyes to PCR, together with a suitable instrument for real-time, online quantification of PCR products during amplification has led to the development of kinetic PCR or quantitative PCR Quantitative PCR (QPC) measures PCR: Basic Principles 15 PCR product accumulation during... Molecular Biology, Vol 192: PCR Cloning Protocols, 2nd Edition Edited by: B.-Y Chen and H W Janes © Humana Press Inc., Totowa, NJ 3 4 Kolmodin and Birch 1.3 PCR Process (see Note 1) The PCR process requires a repetitive series of the three fundamental steps that defines one PCR cycle: double-stranded DNA template denaturation, annealing of two oligonucleotide primers to the single-stranded template, and... protein sequence file Protein.txt (see Table 5) (see Note 4) PCR Primer Design 19 2 Computer Programs for PCR Primer Design and Analysis Bing-Yuan Chen, Harry W Janes, and Steve Chen 1 Introduction 1.1 Core Parameters in Primer Design 1.1.1 Tm, Primer Length, and GC Content (GC %) Heat will separate or “melt” double-stranded DNA into single-stranded DNA by disrupting its hydrogen bonds Tm (melting temperature)... is reversed by a high temperature, pre -PCR incubation (e.g., 95°C for >10 min) The pre -PCR incuba- 6 Kolmodin and Birch tion links directly to the denaturation step of the first PCR cycle So, the reaction mixture never sees active polymerase below the optimal primer annealing temperature If the pre -PCR incubation is omitted, the modification is reversed during the PCR cycling, and polymerase activity... Tris-buffered PCR at 92°–95°C (Tris-Cl formulated to pH 8.3 at 25°C drops below pH 7.0 above 90°C) AmpliTaq Gold is formulated to perform the same as 5 U/µL AmpliTaq DNA polymerase Therefore, a hot start can be added to most PCRs optimized with AmpliTaq DNA polymerase by substituting AmpliTaq Gold and adding a 10-min, 95°C, pre -PCR, activation step The same results can be achieved without the pre -PCR. .. pH 7.0 with NaOH 4 Primer 1: SK145 25 mM in 10 mM Tris-HCl, pH 8.3 at room temperature Sequence: 5'-AGTGGGGGGACATCAAGCAGCCATGCAAAT-3' 5 Primer 2: SK431 25 mM in 10 mM Tris-HCl, pH 8.3 at room temperature Sequence: 5'-TGCTATGTCAGTTCCCCTTGGTTCTCT-3' 6 AmpErase ® UNG: Uracil-N-glycosylase, 1.0 U/mL pH 8.3 at room temperature in 150 mM NaCl, 30 mM Tris-HCl, pH 7.5 at room temperature, 10 mM ethylenediaminetetraacetic... the DNA sequence is entirely random (which may not be true), the chance of finding an A, G, C, From: Methods in Molecular Biology, Vol 192: PCR Cloning Protocols, 2nd Edition Edited by: B.-Y Chen and H W Janes © Humana Press Inc., Totowa, NJ 19 20 Chen, Janes, and Chen or T in any given DNA sequence is one quarter (1/41), so a 16 base primer will statistically occur only once in every 416 bases, or about... sequenced, and analyzed as usual Pretreatment of each PCR reaction with uracil-N glycosylase (UNG), which catalyzes the removal of uracil from single- and double- PCR: Basic Principles 5 stranded DNA, will destroy any PCR product carried over from previous reactions, leaving the native T-containing sample ready for amplification (10) 1.5 Hot Start PCR is conceptualized as a process that begins when thermal... the DNA sequence is entirely random (which may not be true), the chance of finding an A, G, C, From: Methods in Molecular Biology, Vol 192: PCR Cloning Protocols, 2nd Edition Edited by: B.-Y Chen and H W Janes © Humana Press Inc., Totowa, NJ 19 20 Chen, Janes, and Chen or T in any given DNA sequence is one quarter (1/41), so a 16 base primer will statistically occur only once in every 416 bases, or about . Programs for PCR Primer Design and Analysis Bing-Yuan Chen, Harry W. Janes, and Steve Chen 19 3Single-Step PCR Optimization Using Touchdown and Stepdown PCR Programming Kenneth H. Roux 31 4XL PCR Amplification. Y TM John M. Walker, S ERIES E DITOR Humana Press Totowa, New Jersey PCR Cloning Protocols Second Edition Edited by Bing-Yuan Chen and Harry W. Janes Rutgers University, New Brunswick, NJ M E. ISBN 0-8 960 3-9 6 9-2 (hb : alk. paper) ISBN 0-8 960 3-9 7 3-0 (comb. : alk. paper) 1. Molecular cloning Laboratory manuals. 2. Polymerase chain reaction Laboratory manuals. I. Chen, Bing-Yuan. II. Janes,