PCR Product Clean-up General Information
Polymerase chain reaction (PCR) is a technique in molecular biology that is used to amplify copies from a piece of DNA to generate millions of copies of a particular DA sequence. Cleaning up PCR products may not be needed before the DNA amplification, however it becomes significant during the next step. PCR product clean-up is an important step to avoid PCR primers and Taq polymerase – types of high resistant bacteria that can potentially affect the results of sequencing PCR products and TA cloning.
If PCR products are not cleaned after modification, the extra primers (e.g. excess dNTPs, unincorporated primers, and non-specific PCR products) can extend the PCR sequence resulting in the generation of an extra sequence of DNA fragments, thereby, making sequencing data interpretation more difficult.
There are several ways to clean up PCR product.
- Using a low melting point, you may run the PCR product on an agarose gel. This method is recommended for PCR products that do not have single clean band. Once done, you may expose the PCR under UV light and remove the band using a razor. You can extract the DNA from the gel by electroelution or gel extraction methods using buffers and columns.
- For a faster and easier PCR clean-up, you may opt to use magnetic beads. Magnetic beads allow the PCR product to bind and wash away extra primers. The PCR product is then eluted from the beads through magnetization.
- You may also use a spin column technique to quickly purify the PCR products. This method allows the PCR to remain in the column during slow centrifuge spins while washing off the extra primers.
PCR Clean- up Protocol
- Make sure that your PCR product does not contain any double bands. Upon visualizing the VCR product, the ones that show brighter on the gel has strong amplification, which means that they can sequence better.
- Prepare enough 200µL strip tubes for each of the PCR product and write a label on each of their sides including the date and the initial. Do not sequence the samples that did not amplify.
- Add 5µL of PCR product to each of the tube by using a high DNA pipette. This provides more templates for two sequencing reactions. You can scale up the measurement if you need more templates.
- Create a master-mix of 5µL shrimp alkaline phosphates (SAP) and 5µL exonuclease (EXO) using a NO DNA pipette. Mix well together by pipetting down and up.
- Add 1µL of the master mix foe every tube and then spin the tubes at high centrifuge.
- Incubate the samples by setting the thermocycler to 37°C for 30 minutes and then cool at 25°C.
- Once done, the samples are ready for sequencing depending on how bright the bands were.