The products of a polymerase chain reaction
commonly contain a mixture of products due to the amplification of unwanted or non-specific PCR fragments. This results in a range of bands on a gel often referred to as a "ladder" or "smear". Only one of these bands represents the desired DNA fragment. It can be difficult to find the correct PCR parameters with which to obtain the correct band in a pure state while still maintaining yield. Attempting to purify the band by cloning all the reaction products and then probing the library for the correct DNA can be very tedious and time-consuming.
A simple "core sampling" procedure can be used to isolate the required fragment from a mixture of PCR products. This post-PCR procedure involves "coring" an agarose sample out of a gel and using the sample as template in another round of PCR. This is a good "work-around" to obtain unique bands from a background of a large number of unwanted DNA fragments. Having a visible band of the required size is recommended although it is still possible to attempt the technique on "right-sized" invisible bands if a visible band cannot be achieved.
The full protocol for this PCR technique is available on-line at Core-Sampling a PCR Band
- PCR Troubleshooting: The Essential Guide
- Real-Time PCR: Current Technology and Applications
- Real-Time PCR in Microbiology: From Diagnosis to Characterization
- PCR Books
Labels: core sampling, PCR cloning, protocols, reamplification