The PCR and HRM master mixes were prepared to yield final working concentrations of 1.66 mM (NH4)2SO4, 6.7 mM tris (pH 8.8), 2.7 mM MgCl2, 1 mM β-mercaptoethanol, 300 nM primers (IDT; table S1), 200 μM dNTPs (Thermo Fisher Scientific), 1× ROX (Thermo Fisher Scientific), Platinum Taq DNA polymerase (0.04 U/μl) (Thermo Fisher Scientific), bovine serum albumin (New England BioLabs) (1 mg/ml), 0.01% Tween 20 (Sigma-Aldrich), and 1× EvaGreen (Biotium). This master mix was drawn into microcentrifuge tubing (Cole-Parmer) using a syringe. Because of the desiccation, a negative pressure differential existed inside the microfluidic chip seal. When the needle of the tubing punctured the seal, the sample was drawn into the wells. Partitioning fluid, consisting of 5 g of silicone oil and 1 g of PDMS (10:1), was then pressurized through the channels. Because of surface tension, the fluid did not enter the wells and served to isolate the reaction chambers and digitize the sample throughout the 4096 wells. During PCR, the partitioning fluid was pressurized at one end and sealed at the other. Furthermore, the PDMS in the oil solidified over the course of the PCR, producing a permanent barrier.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.