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Analysis of PCR Products

Learn How to Analyze PCR Products and PCR Reactions

Agarose Gel Electrophoresis Analysis of PCR Products

To quickly and easily analyze and resolve PCR products, a 3% NuSieve can be used GTG agarose (FMC Bioproducts, Rockland, ME) and 1% Seakem GTG agarose (FMC Bioproducts) gel run in either TBE (89 mM Tris-borate, 2 mM EDTA) or TAE (40 mM Tris-acetate, 2 mM EDTA, pH approx 8.5). The resolved DNA bands are detected by staining the gels with either approx 0.5 μg/mL of ethidium bromide, followed by destaining with water or SYBR® Green 1 (Molecular Probes Inc., Eugene, OR) and finally photographed under UV illumination. Use a 123-basepair (bp) or 1-kilobasepair (kbp) ladder as a convenient marker for size estimates of the products.

Other Analytical Methods to Analyze PCR Products

There are a variety of other detection methods available for PCR product analysis, such as ethidium bromide-stained 8–10% polyacrylamide gels run in TBE buffer, Southern gels or dot/blots, subcloning and direct sequencing, HPLC analysis, and the use of 96-well microplates, to name a few. The reverse dot-blot method combines PCR amplification with nonradioactive detection. 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 product accumulation during the exponential phase of the reaction and before amplification becomes vulnerable, i.e., when reagents become limited. The ABI Prism 7700 (Applied Biosystems) and the LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) are integrated fluorescent detection devices that allow fluorescence monitoring either continuously or once per cycle. These instruments can also characterize PCR products by their melting characteristics, e.g., to discriminate singlebase mutations from a wild-type sequence. The recently designed Mx4000™ Multiplex Quantitative PCR System (Strategene, La Jolla, CA) can generate and analyze data for multiple fluorescent real-time QPCR assays.

Can't Find the Solution to your PCR Problem? Make sure you visit the PCR Forum for PCR troubleshooting and solutions to common PCR problems. You can also post questions to other researchers by simply registering first and then clicking on Post New Thread button in the forum, or simply clicking the question mark below!

PCR Forum Topics

loading dye Has anybody ever come across a loading dye that ran at a different molecular size on an agarose gel than expected? Why is this happening?
Cresol Red/Sucrose Hi. Does anyone have experience using cresol red/sucrose as loading dye added to the reaction BEFORE PCR? Does cresol red/sucrose have to have a...
Amplification won't work! I'm trying to amplify a ~200bp segment from a ~4kb plasmid. A postdoc in my lab previously amplified the segment and gave me a stock of gel-extracted...
Any ideas why my PCR amplification won't work? I'm trying to amplify a ~200bp segment from a ~4kb plasmid. A postdoc in my lab previously amplified the segment and gave me a stock of gel-extracted...
Heated lid on RFLP: is it ok? I'm doing three different RFLPs with three different restriction enzymes using a single amplicon, each RFLP in different tubes of course as each...
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