Generally for multiplexing you would check all of your primers for compatibility with the other primers to minimise primer dimer but if you are only duplexing then I would not change any volumrs or reagent concentrations,add both primer sets and run the pcr at the temperature that works for the lowest annealing primer pair. All of the reagents in PCR are in large excess so no changes are needed and the amount of dna template should also remain the same as for one pcr. If you get a primer dimer then reduce the primer concentrations and use a hot start enzyme but if you already have the primers working then just give it a try

I wouldn't waste my time optimizing a multiplex reaction unless I really needed to create one. There are so many ways it can go wrong (primers have different annealing temps, one product is preferentially amplified over the other, products are so close in size it's hard to resolve them, etc.). I'd set up different reactions for each gene of interest.

But if it's for something like clinical diagnostics where time and space very limited, then I'd try it with primers that I know give robust amplifications at a range of annealing temps. As Paul Rutland says, use the lower of the two annealing temps and also use the longer of the two extension times.

Good luck!

As per your Kurushanthan Vithujan discussion and question. you have already standardized simplex PCR assay for individual bacteria and you want to multplex. You can start with the following points

1. check for common annealing temperature giving sharp bands by using Gradient simplex PCR assay

2. Just double the concentration of each and every thing in reaction mixture you have added separately for individual bacteria and make a reaction mixture of double volume. For example if you are having standardized reaction for 25 microlitre then you should make 50 microlitre reaction mixture and run with positive controls.

3. If this work then standardize the multiplex by using multiplex gradient and check for the best

4. check the sensitivity and specificity of multiplex PCR assay standardized

you can check the appropriate primer concentration by checker board method or if you get good band by 3. step then you no need to change anything but if one of the band is faint and another sharp you can alter primer concentration.

for more help you can follow my article one specially on multiplexing E.coli, staphylococcus aureus, streptococcus agalactiae and Streptococcus dysgalatiae Article Detection of major mastitis pathogens by multiplex polymeras...

Article End Point Multiplex PCR for Diagnosis of Haemoprotozoan Dise...