1. Multiplex PCR is a valuable tool in many biological studies but it is a multifaceted procedure that has to be planned and optimised thoroughly to achieve robust and meaningful results. In particular, primer concentrations have to be adjusted to assure an even amplification of all targeted DNA fragments. Until now, total DNA extracts were used for balancing primer efficiencies; however, the applicability for comparisons between taxa or different multiple-copy genes was limited owing to the unknown number of template molecules present per total DNA.

2. Based on a multiplex system developed to track trophic interactions in high Alpine arthropods, we demonstrate a fast and easy way of generating standardised DNA templates. These were then used to balance the amplification success for the different targets and to subsequently determine the sensitivity of each primer pair in the multiplex PCR.

3. In the current multiplex assay, this approach led to an even amplification success for all seven targeted DNA fragments. Using this balanced multiplex PCR, methodological bias owing to variation in primer efficiency will be avoided when analysing field-derived samples.

4. The approach outlined here allows comparing multiplex PCR sensitivity, independent of the investigated species, genome size or the targeted genes. The application of standardised DNA templates not only makes it possible to optimise primer efficiency within a given multiplex PCR, but it also offers to adjust and/or to compare the sensitivity between different assays. Along with other factors that influence the success of multiplex reactions, and which we discuss here in relation to the presented detection system, the adoption of this approach will allow for direct comparison of multiplex PCR data between systems and studies, enhancing the utility of this assay type.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3573865/

Sure, I realize how complicated multiplex-PCR is. I just re-checked my results from 4.07 until now. Pan-derm primers were thaw and freezed every day for 6 days, maybe that is the reason the bands are fainter and fainter - I wil run new stock tomorrow.

Another possibility is if you dissolved the primers in water then it absorbs CO2 from the air and the primer depurinates. TE is better with alkaline buffering and EDTA to chelate divalent ions so that nucleases can not work but I also think that thawing too often will degrade primers

I think the product size is also important. is the faint band larger or smaller in length than the other strong bands?

this will help decide what you should do next

if shorter one is weak you can do the following

Increase KCl (buffer) concentration to 1.2x-2x, but keep

MgCl2 concentration at 1.5-2mM

Decrease denaturing time

Decrease annealing time and temperature

Decrease extension time and temperature

Increase amount of primers for the "weak" loci while

decreasing the amount for the "strong" loci.

Add adjuvants. Best, use BSA (0.1 to 0.8 μg/μL final

concentration). You can also try 5% (v/v, final

concentration) DMSO or glycerol

Combine some/all of the above

but if large loci is weak you can

Decrease KCl (buffer) concentration to 0.7-0.8x, but keep

MgCl2 concentration at 1.5-2mM

Increase MgCl2 concentration up to 3-4.5 mM but keep

dNTP concentration constant.

Increase denaturing time

Increase annealing time

Decrease annealing temperature

Increase extension time and temperature

Increase amount of primers for the "weak" loci while

decreasing the amount for the "strong" loci

Add adjuvants. Best, use BSA (0.1 to 0.8 μg/μL final

concentration). You can also try 5% (v/v, final

concentration) DMSO or glycerol

Combine some/all of the above

and here is a useful link

http://www.mudphudder.com/wp-content/uploads/2009/03/pcr-troubleshooting.pdf

Hi, thanks for advices.

I ran new stock yesterday, it's getting better, but still some products are faint (maybe I should manipulate with sample volume, I add 2.0ul of template to 8 ul of Mastermix).

I tried DMSO, but without good results, so I ordered a anti-PCR inhibitor, maybe that will do.

Faint products are 366 bp, but pan-candida products are about 300 bp, the difference is not so big, but recognizable in electrophoresis, S. brevicaulis product is 223 bp.