1. 2 VARIANTS OF PCR APPLICATIONS OF PCR MECHANICS OF PCR WHAT IS PCR? PRIMER DESIGN.
How to select a reliable PCR machine
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The Fashion of Thermal Cyclers Years ago when I was a post doctoral fellow, I did PCR with three water baths, three timers. Quickly, all kind of thermal cyclers come to market. And the prices are constantly going down! Our first lab paid over $10,000 for a toaster oven type, the cheapest we could afford at that time. Now you can buy a new fancy one for less than $5K How do different PCR machines stack up? Any scientific data? Yes, here is a short abstract: Interlaboratory Study on Thermal Cycler Performance in Controlled PCR and Random Amplified Polymorphic DNA Analyses Ginny C. Saunders 1 , Juliet Dukes 1 , Helen C. Parkes 1,a and Johanne H. Cornett 1 1 Life Sciences Research, LGC Ltd., Queens Road, Teddington, Middlesex, TW11 0LY, United Kingdom. a Author for correspondence. Fax 44-020-89432767; [email protected]. Background: Intercomparisons of PCR-based data between laboratories require an assurance of assay reproducibility. We performed an interlaboratory study to investigate the contribution made by a variety of thermal cycler s to PCR performance as measured by interblock reproducibility and intrablock repeatability. Methods: Two standardized assays designed to minimize the introduction of non- thermal - cycler - dependent variations were evaluated by 18 laboratories in the United Kingdom, using 33 thermal cycler s of various makes and models. We used a single-product (590 bp) PCR, established in our laboratory as a robust and specific reaction. The second reaction, a multiproduct random amplified polymorphic DNA (RAPD) PCR, was known to be more susceptible to small changes in block temperature and was therefore considered a way of assessing block uniformity with respect to temperature. Assay repeatability data were analyzed with respect to temperature calibration status, the type of temperature control mechanism, thermal cycler age, and the presence of oil overlay or heated lid systems. Results: All (100%) of the laboratories produced the correct target for the single-product PCR assay, although substantial variation in yield in replicate reactions was observed in 9.4% of these. The RAPD reaction generated results that varied extensively both within the same block and between different thermal cycler s. For eight replicates of a positive sample, 88% intrablock repeatability was demonstrated in calibrated thermal cycler s, which decreased to 63% in noncalibrated instruments. Conclusions: Irrespective of the make and model of thermal cycler , temperature-calibrated instruments consistently generated more repeatable RAPD data than noncalibrated instruments. Guidelines are offered on optimizing and monitoring thermal cycler performance. As you can see, the light flash type offer more variations. Any other types, all the stuff pretty much produced very similar results.
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Transcript of How to select a reliable PCR machine
The Fashion of Thermal Cyclers
Years ago when I was a post doctoral fellow, I did PCR with three water baths, three timers. Quickly, all kind of thermal cyclers come to market. And the prices are constantly going down! Our first lab paid over $10,000 for a toaster oven type, the cheapest we could afford at that time. Now you can buy a new fancy one for less than $5K
How do different PCR machines stack up? Any scientific data? Yes, here is a short abstract:
Interlaboratory Study on Thermal Cycler Performance in Controlled PCR and Random Amplified Polymorphic DNA Analyses
Ginny C. Saunders1, Juliet Dukes1, Helen C. Parkes1,a and Johanne H. Cornett1
1 Life Sciences Research, LGC Ltd., Queens Road, Teddington, Middlesex, TW11 0LY, United Kingdom.a Author for correspondence. Fax 44-020-89432767; [email protected].
Background: Intercomparisons of PCR-based data between laboratories require an assurance of assay reproducibility. We performed an interlaboratory study to investigate the contribution made by a variety of thermal cyclers to PCR performance as measured by interblock reproducibility and intrablock repeatability.
Methods: Two standardized assays designed to minimize the introduction of non-thermal-cycler-dependent variations were evaluated by 18 laboratories in the United Kingdom, using 33 thermal cyclers of various makes and models. We used a single-product (590 bp) PCR, established in our laboratory as a robust and specific reaction. The second reaction, a multiproduct random amplified polymorphic DNA (RAPD) PCR, was known to be more susceptible to small changes in block temperature and was therefore considered a way of assessing block uniformity with respect to temperature. Assay repeatability data were analyzed with respect to temperature calibration status, the type of temperature control mechanism, thermal cycler age, and the presence of oil overlay or heated lid systems.
Results: All (100%) of the laboratories produced the correct target for the single-product PCR assay, although substantial variation in yield in replicate reactions was observed in 9.4% of these. The RAPD reaction generated results that varied extensively both within the same block and between different thermal cyclers. For eight replicates of a positive sample, 88% intrablock repeatability was demonstrated in calibrated thermal cyclers, which decreased to 63% in noncalibrated instruments.
Conclusions: Irrespective of the make and model of thermal cycler, temperature-calibrated instruments consistently generated more repeatable RAPD data than noncalibrated instruments. Guidelines are offered on optimizing and monitoring thermal cycler performance.
As you can see, the light flash type offer more variations. Any other types, all the stuff pretty much produced very similar results.
The key for selecting a reliable thermal cycler unit is the service and warranty. ThermoExp500 system comes with two year standard warranty!
