EricChu:RepliPHI MDA protocol for microfluidic device

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RephiPHI MDA Protocol for single cell MDA microfluidic device[edit]

Device Preparation

  1. Load all valve lines with autoclaved H2O
  2. 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

  1. Create master mix (w/ 1% Tween20)
    1. 2ul 10x buffer
    2. 5ul 200uM N6 primers
    3. 0.8ul 25mM dNTPs
    4. 2ul Phi29 polymerase
    5. 2ul 8% Tween20
    6. 4.2ul H20
  2. Prepare ALS: 69.5ul H20 + 10ul DTT(1M) + 8ul KOH (5M) + 12.5ul 8% Tween20
  3. 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!!!

  1. Load cells
  2. Capture a single cell near the center of the capture chamber
  3. Push single cell into the 1st ring with ALS
  4. Mix for 5min
  5. Push 1st ring content under low airflow (~1psi from red air inlet) into 2nd ring
  6. Fill the 2nd ring with NS
  7. Mix for 5min
  8. Push 2nd ring content under low airflow (~1psi from blue air inlet) into 24-compartments
  9. Remove airflow entirely before separating the compartments
  10. Load the MDA chambers with MDA master mix
  11. 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
  12. Load TE buffer with 0.5% Tween20, and collect about 5ul (for sequencing) or 10ul (for PCR quantification) from each outlet
  13. Heat deactivate polymerase at 65C for 5min


Device Specification

  1. Flow channel (blue layer) width : 120um, height : 12um
  2. Flow channel (red layer) width : 200um, height : 18um

File:3rdGenDevice.jpg