Articles

The first article in this series looked at how thermal cycler manufacturers have improved the lives of PCRers. This time, we'll look at various other time-saving reagents, technologies, and even some gadgets—from morphing microplates to six minute gels.

Ten Minutes ‘Til Show Time
Six-Minute Gel
Fastest Gun (That's Pipette Gun) in the West
Taking the Gel Out of PCR

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Ten Minutes ‘Til Show Time (Back to Top)
While there are DNA preparation machines and robots that can take your place at the lab bench in preparing templates for PCR, Pierce Chemical has a new product for those doing their own preps. Its Lyse-N-Go PCR Reagent is all you need for making template, eliminating the need for lengthy DNA purification protocols. The proprietary buffered lysis reagent releases DNA and inactivates nucleases in the sample with a brief heating step. Since it's compatible with most commercially available Taq polymerases and other thermal stable enzymes, you just lyse your cells, and you're ready for PCR; or, as the name says, just lyse and go. In certain cases, you can even do the PCR in the original tube, which not only saves you the time of transferring samples but also reduces the risk of contamination. Lyse-N-Go can be used with plasmid or genomic templates, bacterial and yeast clones, cultured mammalian cells, blood, and plant leaves.

PCR amplification of DNA from various sources: lanes 2-4: urate oxidase gene from E. coli on a plasmid; lanes 5-7: b-galactosidase gene on a plasmid; lanes 8-10: streptavidin gene from yeast genomic DNA; lanes 11-13: ACT1 gene from a yeast clone; lane 14-16: histone 4 gene from Hela cells.

For more information: Pierce Chemical Co., 3747 North Meridian Rd., Rockford, IL 61105. Tel: 800-874-3723.

Six-Minute Gel (Back to Top)
And then there's the six-minute gel. A company called 6mgel.com has fashioned a gel apparatus than combines high voltage with multi-rows to jack up the number of samples you can run per unit time. It's 6M10 unit holds 96 or 148 samples in four rows, is compatible with multi-channel pipettes (an important consideration for preserving one's wrist), and has a cooling system that lets you run the gel at high voltage, between 250 and 350 volts. Not enough capacity? The king-size gel system (18x14 cm) holds 220 samples, and takes only a couple of minutes more to run.

The key here is cooling. In conventional gel devices, gels are typically overlaid with running buffer to provide cooling, but the high conductivity of the running buffer creates a large current. This in turn generates massive heat in addition to heat from the gel itself, and can cause distortion and, in the extreme, melting of gel.

To overcome this problem, the 6M10 unit uses water (deionized, distilled, or even tap water) as a coolant to absorb heat from the gel and as an insulator to reduce electric current. Gels can be run under high voltage, up to 350 volts, which accelerates migration and reduces run times.

6mgel.com is a factory online outlet of Biokey American Instruments Inc.

For more information: Biokey American Instruments Inc., 18510 SW Honeywood Dr., Beaverton, OR 97006. Tel: 503-649-5275.

Fastest Gun (That's Pipette Gun) in the West (Back to Top)
Embi Tec can get you through the loading woes faster, with its Multi Channel Ultra Micro SequaPipette and the Round Filter SequaStrip. Useful at all stages of PCR, the Strip, exclusive to Embi Tec, lets you pipette 8 samples at a time—an entire 96 or 384 well plate can be pipetted in minutes. You can also use the filter strips to load high throughput gels in a fraction of the time.

Embi Tec also sells pre-cast gels in a dizzying array of agarose concentrations, sizes, and well configurations—from 9 wells all the way to 108 wells. Designed to work with multi-channel pipettors, these gels save time both in the preparation and loading steps.

Here's a novel idea—the morphing microplate. Embi Tec offers a device that causes microplates to expand and contract. This handy device, called the Morph Sequencing Gel Loader, alters the well spacing on the plate such that spacing on microplates that fit in thermal cyclers matches that of a 96-well gel. No need to transfer samples, or do repetitive pipetting with this device in hand.

For more information: Embi Tec, 7738 Arjons Dr., San Diego, CA 92126. Tel: 800-255-1777.

Taking the Gel Out of PCR (Back to Top)
A new generation of PCR detection systems frees you of gels altogether. This approach to PCR product detection uses microwell plate formats and various, mostly non-isotopic labeling methods. It's good for high throughput—you can do 96 assays at once using plate readers or imagers—as well as your health. No isotopes, no ethidium bromide.

Originally developed for light detection, with a horse radish peroxidase detection system, Chemicon has now incorporated bioluminescence in the form of the recombinant photoprotein Aqualite into its ChemFLASH Detection System, an immunoassay format for universal amplicon detection. Aqualite increases the sensitivity and the dynamic range of the assay significantly. Chemicon has a number of ready-to-use kits using this technology, and in addition will custom conjugate AquaLite to antibodies or oligos that can be used as probes.

The ChemFlash system detects biotinylated amplified DNA in a two step process. The PCR reaction is done with one biotinylated primer, after which the product is captured on the plate via a specific capture probe (user-supplied). Streptavidin-conjugated Aqualite is then added, which is induced to flash by adding calcium as a trigger. This flash on demand provides low backgrounds and a six log dynamic range.

For more information: Chemicon International Inc., 28835 Single Oak Dr., Temecula, CA 92590. Tel: 800-437-7500.

And if it's a particular PCR product you're after, Synthetic Genetics offers a line of 96-well oligo plate assays for the detection of a number of clinically important pathogens, including Enterovirus, HBV, EBV, HCMV, VZV, HBV, Borrelia burgorferi, Bordetella Pertussis, Mycoplasma Pneumonia, and HHV-6. Synthetic Genetics uses a proprietary method to attach a capture probe at high density to a 96-well microtiter plate, which is sensitive enough to detect a single copy of amplified DNA in a reverse dot blot format.

The target is amplified using biotinylated primers that come with the system. The amplified product is then transferred directly to the plate, where it binds to the capture probe. Streptavidin conjugate is then added and the plate is developed with a TMB substrate. The entire reaction takes less than 90 minutes and can be read on a standard plate reader.

For more informatin: Synthetic Genetics, 3347 Industrial Ct., San Diego, CA 92121. Tel: 800-562-5544.

Next up: A universal combination amplification and detection system from Intergen (Purchase, NY).

By Laura DeFrancesco