I usually do my PCR with three primer pairs (pan-dermatophytes, pan-candida, and S.brevicaulis), I've noticed most of products of pan-dermatophytes PCR are very faint. I use almost the highest concentration of pan-dermatophytes primers (0,5 uM), for other primers I use 0,3 uM. Final extension is 10 minutes. There are no cross-reactions between primers. Other products are OK. Annealling T is 60oC for 30s, it works for the rest of the primers.
What can I do better? Increase annealling time, decrease annealling temperature? Should I order new primers? I ordered pan-dermatophytes in 2012, but the stock was thawed max 10-15 times to make some new primers concentrations.
Also, maybe some of you know how to make an internal control for PCR?
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
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