The PCR Blog: The Polymerase Chain Reaction

An expert guide to PCR troubleshooting. The online guide covers problems with the template DNA, inadequate dNTPs, primer concentration, Taq concentration, Mg concentration, and KCl concentration. Most PCR problems are caused by one or other of these variables.

Find out the theory and possible solutions to these problems at PCR troubleshooting.

Labels: trouble shooting, Troubleshooting

Zuo and Rabie describe a new one-step "Quick Assemble" method of precisely and simultaneously joining multiple DNA fragments followed by intramolecular ligation. This technique of plasmid assembly and circularization can be used to construct new plasmids with any promoter, resistance gene marker, restriction site, or tag.

The new 2-day PCR-based method can be used to generate both circularized and linear final assembled products. The method makes the synthesis and assembly of large molecules in a quick and efficient way, while circumventing the use of DNA ligases. The ligase-free method also offers an additional improvement over the combination of long PCR and overlap extension PCR, and is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as multiple site-direct mutagenesis and whole-DNA library gene shuffling.

from Zuo and Rabie in Curr. Issues Mol. Biol. 12: 11-16

Further reading: Assembly of DNA fragments into circular constructs

Labels: cloning, PCR cloning

The polymerase chain reaction (PCR) has been widely used for amplification of DNA sequences. The PCR technique can be coupled with a variety of detection assays for the identification of a wide range of DNA targets. Reducing PCR to the microchip level is of interest for portable detection technologies and high-throughput, massively parallel analytical systems. The basic process of creating a miniaturized, microfluidic PCR chip and a simple method for thermally cycling this microchip during PCR amplification have been described (Herold and Rasooly, 2009. Lab-on-a-Chip Technology. ISBN: 978-1-904455-47-9) These methods can be broadly applied to a variety of microchip architectures, materials, and downstream analytical methods.

Labels: PCR chips, PCR on a chip

A comprehensive list of qPCR Web Resources including qPCR machine manufacturers' websites, PCR web resources, and PCR news groups.

from Julie Logan, Kirstin Edwards and Nick Saunders in Real-Time PCR: Current Technology and Applications

Further reading: qPCR Web Resources

Labels: qPCR, real-time pcr

In weighing up the pros and cons of the different real-time PCR platforms or qPCR machines for your laboratory, factors to consider include: supported chemistries; multiplex capability for that chemistry; throughput; flexibility; format; easy-of-use and robust software package; reproducibility; speed; size; technical support; customer support and not least the cost, not only of the initial equipment outlay and servicing but also the associated cost of consumables and reagents.

The following qPCR machines are compared for various features to help you decide which instrument is most suitable for your needs.

• Applied Biosystems: ABI 7300, ABI 7500, ABI 7500 Fast, ABI 7900 Fast HT with automation accessory, ABI StepOne
• Roche: LightCycler 480, LightCycler 1.5, LightCycler 2.0
• Stratagene: Mx4000, Mx3000P, Mx3005P
• Cepheid: SmartCycler
• Corbett: Rotor-Gene 6000
• Eppendorf: Mastercycler ep realplex
• BioRad: MiniOpticon, MyiQ, Opticon2, Chromo4, iQ5

read more ...

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Labels: qPCR, qPCR instruments, qPCR machines, real time PCR machines

In weighing up the pros and cons of the different platforms for your laboratory, factors to consider include: supported chemistries; multiplex capability for that chemistry; throughput; flexibility; format; easy-of-use and robust software package; reproducibility; speed; size; technical support; customer support and not least the cost, not only of the initial equipment outlay and servicing but also the associated cost of consumables and reagents. It is also possible to 'try before you buy', most companies will provide a loan machine. It is wise to test a few of these once you have narrowed down your choice. User experiences should not be overlooked and there are now a number of useful websites and news groups where you can address you questions and queries.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Labels: ease-of-use, flexibility, format, multiplex, pros and cons, reproducibility, size, software, speed, supported chemistries, technical support, throughput, user experiences

Newly introduced qPCR machines include the Cepheid GeneXpert and Enigma Diagnostics Enigma ML instruments, which combine automated sample preparation and real-time PCR into a single integrated compact bench top instrument, which opens new avenues for real-time PCR in field-based and point-of-care environments. At the other end of the scale is the introduction of nanoliter high throughput quantitative PCR. The Biotrove OpenArray system allows 3072 33nl reactions on a microscope slide array format, which equates to a single operator performing 27,000 qPCR reactions in a working day. Another interesting development on the horizon is ultra fast real-time PCR chips capable of performing 40 cycles in under six minutes.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

1. Real-Time PCR: Current Technology and Applications
2. Real-Time PCR in Microbiology: From Diagnosis to Characterization
3. PCR Troubleshooting: The Essential Guide
4. PCR Books

Labels: Biotrove OpenArray, Cepheid GeneXpert, Enigma Diagnostics, nanoliter high throughput qPCR, PCR chips