First, to prepare a primer mix at 10µM of concentration, you have to start with a mother primer solution of 100µM. e.g. if you have a set of 4 primers (two pairs: P.1/P.2, P.3/P.4), and you want to make a 200µl volume of the primer mix, put a 120µl volume of water in a tube and add 20 µL of P.1 at 100µM, 20 µL of P.2 at 100µM, 20 µL of P.3 at 100µM and 20 µL of P.4 at 100µM.

Second, the primer mix volume that you have to add in 50µl PCR reaction volume depends on the reaction final concentration of your primers. e.g. if the final concentration is 0.4µM = 400nM (should be the same for all primers), then you will need to add 2µl from your 10µM primer mix.

The rule is that C.i x V.i = C.f x V.f --> V.i = (0.4 x 50)/10 = 2µl

Hope that helps

I am not an expert in this field, but I am very interested and have researched to find an answer. Could you please review the response below to see if it is correct?

To prepare a Primer-mix for Multiplex PCR (M-PCR), follow these steps:

Primer Design: Design specific primers for each target sequence. Incorporate a universal primer (UP) adapter at the 5′-end of each specific primer to overcome issues of amplification disparity and self-inhibition [1]. Use tools like CODEHOP to design primers that target conserved amino acid sequences, which can help in identifying related gene sequences across different species [2].

Primer Validation: Ensure the designed primers do not have significant cross-interactions. Tools such as MuPlex can be used to screen for primer-pair interactions and optimize the multiplex PCR assay design [4].

Primer Concentration: Prepare each primer at an equimolar concentration to ensure balanced amplification of all targets. A typical concentration for each primer in the mix can be around 0.1-0.5 µM, but this may need to be optimized based on preliminary experiments.

Mix Preparation: Combine all primers in a single tube with a suitable buffer. For example, if you have ten primers, mix them in equal volumes to reach the final desired concentration.

Quality Control: Validate the Primer-mix by running a preliminary PCR with known templates to ensure all targets are amplified efficiently.

Storage: Store the Primer-mix at -20°C to maintain stability over time.

By following these steps, you can prepare an effective Primer-mix for M-PCR, facilitating high-throughput and reliable amplification of multiple targets [1][3][4].

Reference

[1] Xu, W., Zhai, Z., Huang, K., Zhang, N., Yuan, Y., Shang, Y., & Luo, Y. (2012). A Novel Universal Primer-Multiplex-PCR Method with Sequencing Gel Electrophoresis Analysis. PLoS ONE, 7.

[2] Rose, T., Henikoff, J., & Henikoff, S. (2003). CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design. Nucleic acids research, 31 13, 3763-6 .

[3] Xu, Y., Yan, H., Zhang, Y., Jiang, K., Lu, Y., Ren, Y., Wang, H., Wang, S., & Xing, W. (2015). A fully sealed plastic chip for multiplex PCR and its application in bacteria identification.. Lab on a chip, 15 13, 2826-34 .

[4] Rachlin, J., Ding, C., Cantor, C., & Kasif, S. (2005). MuPlex: multi-objective multiplex PCR assay design. Nucleic Acids Research, 33, W544 - W547.

I received some assistance from tlooto.com for this response.

Thank you dear for your comments