PCR Cleanup
PCR Cleanup Background
Generally, the more you initially optimize your PCR, the less will be your need for stringent post-PCR cleanup methods.
If your PCR conditions are well optimized, most of your added primers and dNTP's will be used up during the pcr amplifications, thus there will be only very few interfering factors and it may be possible to carry out direct sequencing. If you are going to sub-clone your PCR fragment into a TOPO cloning vector, then you need to have only one major PCR band visible on your gel. If you have multiple bands, make sure you optimize your PCR conditions first.
Also see PCR product sequencing and optimizing PCR.
PCR products do not need to be purified prior to sequencing however to obtain accurate sequencing data, it is usually recommended to clean up your PCR products after amplification.
If you have added excessive primer amounts or primers are unused, these primers can act as extension primers during sequencing, resulting in the generation of an additional set of dye-labeled sequencing fragments which can make sequencing data interpretation difficult if not impossible.
Post-cleanup PCR prducts should be run on an agarose gel to examine the quality of the purification. If smearing or multiple bands are present, products smear, you will not usually obatin high quality sequence data.
Gel purification of the fragment can be accomplished using a low melting-temperature agarose. You can remove your band under UV light with a razor, and extract the DNA from the slice using electroelution or gel extraction methods using buffers / columns.
PCR Cleanup for Sequencing and Cloning
To remove excess dNTPs, unincorporated primers, and non-specific PCR products you can use several methods.
Qiagen PCR Purification Kit and other PCR cleanup kits.
You can also remove primers and dNTPs by running the PCR products on an agarose gel, cutting the bands out and conducting DNA extraction from the agarose gel using Qiagen Gel Purification Kit.
After PCR Cleanup
After any PCR clean-up method, you should establish the quality and quantity of the PCR product. You can do this by running the samples using agarose gel electrophoreiss along with a DNA concentration marker.
You can also estimate the quantity of DNA using a UV spectrophotometer.