How do I perform PCR by Random priming using enzyme Q5 Hot Start?

Carolina Campos Martínez @Carolina_Campos_Martinez

05 July 2023 1 6K Report

Hi everyone,

I am currently working with very low concentration of RNA and after Retrotranscription, I would like to concentrate cDNA by doing a PCR with the same random primers I have used for cDNA synthesis (50 uM Random Hexamers) and using he Q5 Hot Start polymerase, but I am not quite sure about concentration of random hexamers in PCR final volume, so does anyone know where can I find this kind of information/protocol?

Thank you.

Jakub Knurek

Hi Carolina,

To perform PCR by random priming using Q5 Hot Start polymerase, you can follow a protocol that combines random hexamers with the enzyme. Here are the general steps to guide you:

Determine the concentration of random hexamers: Typically, the concentration of random hexamers used in PCR reactions is in the range of 0.2-2.5 μM. You mentioned that you are using 50 μM random hexamers for cDNA synthesis. For the PCR reaction, you can start with a concentration of 0.2-1 μM of random hexamers. It's advisable to perform optimization experiments to find the optimal concentration for your specific experiment.

Calculate the volumes of each component based on the desired final reaction volume. Generally, a PCR reaction mix includes the following components:Template cDNA: Use the desired concentration of concentrated cDNA obtained from retrotranscription. If necessary, adjust the cDNA concentration before starting the PCR.

Prepare the PCR reaction mix: - Q5 Hot Start DNA Polymerase: Follow the manufacturer's instructions for the recommended amount of enzyme per reaction volume. - Random hexamers: Add the desired concentration of random hexamers to the reaction mix. - dNTPs: Typically, 200-400 μM of each dNTP is used in a PCR reaction. - Buffer: Prepare the PCR reaction buffer according to the manufacturer's instructions for Q5 Hot Start polymerase. - MgCl2: Determine the optimal concentration of MgCl2 for your specific PCR reaction. It is typically in the range of 1-5 mM. Use the manufacturer's guidelines or perform optimization experiments. - Water: Add nuclease-free water to reach the final reaction volume.

Set up the PCR cycling conditions according to the guidelines provided by the manufacturer of the Q5 Hot Start polymerase. The cycling conditions typically include denaturation, annealing, and extension steps. The annealing temperature depends on the melting temperature (Tm) of the random hexamers used and should be optimized for your specific primer and template combination.

Perform the PCR amplification using the optimized cycling conditions. It is recommended to include appropriate positive and negative controls in your PCR experiments.

It's important to note that the exact protocol and optimization steps may vary depending on the specific requirements of your experiment and the manufacturer's recommendations for the Q5 Hot Start polymerase. You can also follow New England BioLabs protocol, avaiable at the link: https://international.neb.com/protocols/2012/08/30/pcr-using-q5-hot-start-high-fidelity-dna-polymerase-m0493

Good luck

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