EricChu:RepliPHI MDA protocol for microfluidic device: Difference between revisions
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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]] | [[File:3rdGenDevice.jpg]] |
Revision as of 00:23, 5 February 2013
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