PCR – reamplification


If PCR product is not strong enough or there are multiple bands, one solution for getting sufficient quantities of the correct PCR product is to resolve out the sub-standard PCR product on a 1% NuSieve agarose, then cut it out with a Blue Tip, and resuspend them in 50 μl of ddH2O in an Eppendorf tube by heating at 55°C or higher until the NuSieve plug dissolves. The resuspended PCR product is then used instead of genomic DNA in a PCR amplification using the same set of PCR primers used to originally generate the sub-standard PCR product. The sub-standard PCR product can also be resolved on a normal 1% agarose gel. In this case, however, the agarose plug should not be allowed to dissolve in the 50 μl of ddH2O since agarose in higher quantities inhibits PCR. The agarose plug should be warmed at 55°C for 10-15 minutes to facilitate the elution of the PCR product, and then the Eppendorf tube should be centrifuged to compact the agarose on the bottom of the Eppendorf. When I reamplifying the PCR product, the annealing temperature should be raised to 55°C or higher (depending on the annealing temperature of the primers), remember that this time the match between the priming sites and the primer is perfect.

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