Yes. Just use different dyes for each primer pair unless the expected sizes of your amplicons for each pair are known and different enough that you can tell them apart with a single dye.

Oh, and be sure that the primers for each target won't dimerize. You can use free web-based tools to check them for dimerization and other issues (Google "check primer dimer").

If the downstream application is just to sequence the amplicons (not to quantify them), then you can use a conventional thermal cycler. Good luck!

Thank you. Maybe I should've been more clear. I will try to do a microsatellite analysis and I'm not really experienced as you can see. I've been instructed to buy primers dyed with either HEX, FAM or NED. From what I understand I must choose which primer to dye with which one of these dies while paying attention to product sizes, as I only have three dyes to choose from, but 5 or more primers. As Mr. Haggard pointed out, I will use a combination of fragment sizes and fluorosence to tell them apart. But, there are so many other variables, I'm a bit confused. For example, apperantly you can dye the primers at 5' or 3'. I don't know what is the difference. Thank you all.

I'd go with 5' labeled oligos. Adding stuff to the 5' end is usually the safest - least likely to interfere with annealing and polymerase binding. In fact, if you need better separation between some of your primer sets, you can add some "nonsense" sequence to the 5' ends of the primers in one set to make the fragments heavier.

Be aware that NED (yellow) can be a bit more difficult to detect. If I was going to multiplex more than one primer pair with the same dye, I'd try to use HEX or FAM for those and put as few as possible on the NED channel. It's not a big deal though, so don't worry.

If you have genome sequence available, you should probably BLAST your primer sequences against it to be more sure that they will only bind to a single target site, or else you can have problems where primers from different pairs can bind and amplify non-target sequences.

One more tip...you only need to label one primer from each pair.

Best of luck!

Thank you Mr. Haggard. The information you've provided is of great value to me! 

All the best!

Dear Burak,

you are unclear on what you want to do?

if it is real time PCR, you need a real time PCR capable machine!

if it is sequencing of amplicons, any Thermocycler will do. however, if you duplex you will never be able to sequence your amplicons, since there is no good way of separating them for sequencing.

yes definitely

Mr. Eck I apologize for not being clear but I gave some more information about my situation a couple of posts below the original. Multiplex pcr I am tasked to do is for microsatellite analysis. So sequencer will analyse only the fragment sizes. I don't imagine I need to do any quantitative analysis during pcr.