Hi everyone.
I am performing PCR in droplets of 20um of diamater and my protocol is a classical PCR protocol, oil HFE 7500 and surfactant is PicoSurf 3%. I leave like 20uL of oil on the bottom of PCR tube and the emulsion on top. I spin dow the emulsion to keep it compact on top of the oli (is this ok? should I kee doing this?)
Protocol:
95 ºC 4 '
95ºC 30''
64ºC 30''
68ºC 2'
(x30)
68 ºC 5 '
12 º C inf
PCR works, nice yield but my problem is the droplet merging. After the incubation I lose like half of the emulsion and the remaining has a high percentage of merged droplets (like, vey big droplets). I tried putting some mineral oil on top of the emulsion and it makes ir a little better but not perfect and it's an option I'd rather not use because it makes my sample very dirty for the following steps.
If anyone has experience with dropelt PCR and has some adivce I would be very grateful.
Thanks a lot in advance
Jorge
РCR in drops is a patented technology. For its implementation, all pure substances, the sequence of operations, etc. are important. Therefore, you need to check everything, and after verification, contact the technology seller for clarification.
I think you need to work with the thingy which is wrapped in that thing. Let me know how it goes!
You need to tune the HLB value of the droplets by adding oil and water-based surfactants. Generally, SPAN 80 / SPAN 85 (oil-based surfactant) and Tween 80 /Tween 85 (water-based surfactant) are utilized to prevent droplet merging.
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