So one of the bread and butter techniques in science is PCR. And while it can be oh so easy, there are a surprising number of subtleties to each of those steps we take for granted. That is, until our PCR doesn’t work and we’re left wondering why. The worst is when your PCR has worked for 3 months and then all of a sudden it doesn’t work anymore and you’re sitting there saying, WTF?!?!?!? Moreover, PCR has gotten increasingly sophisticated with many different variants compared to what it was like when I started. (When I started, RT-PCR was the cutting edge–yes, I’m that old).
Anyway, in an effort to help out with protocols and troubleshooting, I have assembled a number of online resources which I have found to be useful.
PCR Links.com contains general information (on primers, polymerases, inhibitors, additives, optimization and troubleshooting), online books, as well as information (including protocols) on various types of PCR (including AFLP, Alu-PCR, Asymmetric PCR, Colony PCR, DD-PCR, Degenerate PCR, Hot-start PCR, in situ PCR, inverse PCR, Long-PCR, Multiplex PCR, Nested PCR, PCR-ELISA, PCR-RFLP, PCR-SSCP, QC-PCR, RACE, RAPD, real-time PCR, Rep-PCR, RT-PCR, TAIL-PCR, Touchdown PCR and Vectorette PCR).
From the Real-time quantitative PCR resource at the Iowa State University, attached are an-introduction-to-real-time-pcr-qpcr-assay-design-and-optimization and example-of-an-entire-qpcr-setup.
One good general website for PCR protocols is Protocol-Online for PCR (including protocols for everything from standard PCR, RT-PCR and quantitative real-time PCR to more specialized variants of PCR).
From Yale University, check out Octavian Henegariu’s website for PCR for valuable information on protcols and troubleshooting. I have attached pdf printouts from this website on general PCR guide, choosing/designing PCR primers, designing PCR programs, magnesium concentration, dNTP concentrations, taq polymerases and PCR troubleshooting.
From the University of Helsinki, FastPCR–a free software package that has many capabilities including (from the FastPCR website):
• Standard, inverse, long, real-time PCR analysis – identification of the optimal primers for PCR, hybridization, or sequencing;
• Multiplex PCR primers design – fast primer design with a cross-dimer test for high sensitive multiplex PCRs;
• Group-specific PCR primers design – design of universal PCR primers for all sequences (there is no need for a multiple DNA alignment);
• Unique PCR primers design – design of specific PCR primers for each sequence;
• Degenerate PCR – primers design directly on an amino acid sequence;
• Automatically SSR loci detection and direct PCR primers design;
• In silico PCR and probe search – prediction of probable PCR products and search of potential mismatching location of the specified primer(s)/probe (s);
• TaqMan and Molecular Beacons probes and other probes design;
• LUX (self-quenched) primers design for quantitative PCR;
• Self-Reporting DNA/DNA primers for qPCR analysis;
• Primer Secondary structure analysis – self-dimer and cross-dimer primer analyses; primer alignment and melting temperature calculation;
• False priming analysis – (the secondary (non-specific) binding test) primers checking for multiple annealing sites using sequence alignment algorithms;
• Primer quality report – a unique way for PCR efficiency determination;
• Comprehensive primer report – comprehensive pairs and individual primers analysis (Tm and dimer detection);
• Multitask PCR primers and probes design – simultaneously design primers or probe with different parameters and for different targets within the same sequence; interaction different tasks.
• Repeats search: Invert, Direct, Simple and others;
• MITE elements search;
• LTR-retrotransposons search – discovery new LTRs, clustering and whole elements discovery;
• SSR (Simple Sequence Repeat) locus search – two, three, four or five perfect and imperfect core motif
• Clustering sequences (BLAST2 related alignment);
• Sequence alignment – using universal degenerated code;
• Complement, reverse and reverse-complement strand modification;
• Consensus sequence for two or mores sequences;
• Extraction of specific fragments from a larger sequence;
• DNA-to-Protein translation and Protein-to-DNA reverse translation;
• Calculation of the annealing temperature of PCR (for unknown PCR products);
• Database analysis tools;
• Restriction analysis;
• Tools: patterns analysis – CG% content; purine-pyrimidine ration; GC skew (G-C)/(G+C); the melting temperature and linguistic sequence complexity;
• Sequence complexity profile calculation – fast calculation of linguistic sequence complexity of DNA or protein sequences with sliding windows from 10 to 2000 characters (this approach provided an efficient way to detect – SSR loci, introns-exons, S/MARs (Scaffold/Matrix Attachment Regions) or else);
• Pair Sequences Similarity – calculation the similarity for entered sequences in alignment format like MEGA or GCG/MSF and also non alignment raw formatted sequences.