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EricChu:RepliPHI MDA protocol for microfluidic device
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==RephiPHI MDA Protocol for single cell MDA microfluidic device== '''Device Preparation''' #Load all valve lines with autoclaved H2O #15min UV treatment of all tubings, fully assembled device, and components of MDA (ALS, NS, H2O, and MDA buffer) note: Do not use blocking buffer - it will convert the hydrophobic surface to hydrophilic and will cause sample loss '''Reagent Preparation''' #Create master mix (w/ 1% Tween20) ##2ul 10x buffer ##5ul 200uM N6 primers ##0.8ul 25mM dNTPs ##2ul Phi29 polymerase ##2ul 8% Tween20 ##4.2ul H20 #Prepare ALS: 69.5ul H20 + 10ul DTT(1M) + 8ul KOH (5M) + 12.5ul 8% Tween20 #Prepare NS: 87.5ul NS stock + 12.5ul 8% Tween20 where NS stock: 4ml 1MHCl and 6ml 1MTrisHCl '''Device Operation''' Fill each reagent from its own inlet!!! #Load cells #Capture a single cell near the center of the capture chamber #Push single cell into the 1st ring with ALS #Mix for 5min #Push 1st ring content under low airflow (~1psi from red air inlet) into 2nd ring #Fill the 2nd ring with NS #Mix for 5min #Push 2nd ring content under low airflow (~1psi from blue air inlet) into 24-compartments #Remove airflow entirely before separating the compartments #Load the MDA chambers with MDA master mix #Move the device to the TE module for 5 hours reaction at 30C (setting at 32C). Also, empty the MDA branches with air for the entire time #Load TE buffer with 0.5% Tween20, and collect about 5ul (for sequencing) or 10ul (for PCR quantification) from each outlet #Heat deactivate polymerase at 65C for 5min '''Device Specification''' #Flow channel (blue layer) width : 120um, height : 12um #Flow channel (red layer) width : 200um, height : 18um [[File:3rdGenDevice.jpg|600px]]
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