Rapid re-amplification of PCR products purified in low melting point agarose gels.
A rapid, simple method is described for performing sequential amplifications of purified products produced by the PCR. After the initial amplification, an aliquot of the reaction is run on a low melting point agarose gel. A Pasteur pipet is used to punch out a gel plug from the amplified band. The DNA in this plug is then used directly as the template for a second round of amplification. Relatively large amounts of agarose can be tolerated without noticeable effects on amplification. Use of a composite gel made from agarose and linear polyacrylamide increases the ease and utility of this technique. These gels are simple to cast, easier to handle and permit several replicate plugs to be obtained from a single band. This method is well suited to experiments which use "nested" primers to increase the sensitivity and specificity of amplification or any method in which PCR amplification follows DNA purification by electrophoresis in LMP agarose gels.
Zintz
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Department of Anatomy, USUHS, Bethesda, MD 20814-4799.
Beebe
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D004587:Electrophoresis, Agar Gel; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature