Concentration of Eva green with different amplicon sizes in a PCR pool - (Dec/02/2014 )

I've worked in Evagreen for 3 months. I used multiplex PCR which expected amplicon sizes about 150bp, 200bp, and 400bp. Interestingly, with optimum PCR MgCl2 (2.0mM) and 1X Evagreen, it only amplified 2 kinds of product with shorter range amplicon size (150bp and 200bp) while fragment 400bp was not obtained. I've read some paper, they mentioned that using Evagreen 0.5-1X and MgCl2 2.5mM were the optimum conc for long range amplicon work well. I tried it but I did not get the right thing. So, please let me know:

1. What suitable Evagreen and MgCl2 conc to get these products.

2. Should I will increase ext time now I used temp cycle as 40 cycles of (95oC-30s/60oC-30s/70oC-30s)

3. Did we should limit the amplicon length for Evagreen working.

Best regard!

-hthphuong89-

I would first make sure that the primers for the 400 bp amplicon work in the non-multiplexed environment. I'm not sure what you are doing with Evagreen in a multiplex PCR -- you can't be doing a qPCR reaction with more than one product, so why is the evagreen present in the reaction?

-phage434-