We use a manual droplet generation microfluidic device to generate droplet.
We use syber green dye for this purpose (PCR). Is this dye appropriate for ddPCR?
What ratio of DNA, primer and mastermix is ideal for ddPCR?
Is a 96 well plate suitable for fluorescent reading or not?
Hi Amir,
SYBR Green should not be used for ddPCR, it's not compatible with the QX systems of BioRad (check their manual: https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf). EvaGreen on the other hand should be fine, but I would probably suggest using the reagents by BioRad if you use their system.
In terms of MasterMix, you should not use more than 330ng/µl of DNA. In my reactions, I used 10 µl of supermix and added 2 µl of 10 µM of each primer. I don't know how much fluorescent dye you need though, because I used probes in my assay (0.5 µl). I ended up with 23 µl MasterMix reactions (17 µl Mastermix, 5 µl DNA) which I added to a 96 well plate, so that should be fine!
In terms of Droplets, I learned to reject reactions with less than 10.000 droplets. If it's a negative or positive signal will be decided based on your threshold which you should set manually or at least check what your software suggests to you. An excellent paper for this that helped me loads: Article Challenges Using Droplet Digital PCR for Environmental Samples
Good luck!
Dear Amir Asri thank you for your very interesting technical question. I addition to the excellent and rather comprehensive answer provided by Mirjam Seeliger please also have a look at the following useful article in which the use of EvaGreen is described in detail:
Droplet Digital PCR with EvaGreen Assay: Confirmational Analysis of Structural Variants
Article Droplet Digital PCR with EvaGreen Assay: Confirmational Anal...
Also please see the following guide which is freely available as public full text::
Droplet DigitalTM PCR Applications Guide
https://www.gene-quantification.de/bio-rad-ddpcr-app-guide-6407.pdf
(see attached pdf file)
Good luck with your research and best wishes!
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