EricChu:LabNotesMDA/2013-5-20
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Use CoRE Protocol for MDA in Device v7
- Single MEF cell was planned and used in this protocol. The difference in mastermix preparation to the experiment on May17 was the additional dUTP in this CoRE protocol.
- MDA was performed in the device for 15 hours at 30C.
- Since I believe some MEF cells lost the DNA after a few hours in PBS on ice, all experiments will only use freshly trypsinized cells in culture media.
MDA Device v7 Preparation
- Filled all valve lines with water; leaked check (only one valve to close the NS channel was leaking, but it was not important for the functionality of the device)
- At the meantime, 1ul 200uM N6 was mixed to 4ul 0.1xBSA in a PCR tube.
- Loaded N6 and BSA from the outlet of ALS+DNA.
- Allowed 30mins incubation in the closed channels of MDA mastermix, NS, ALS+DNA and 23nl chambers.
- Released the bottom valve of the 23nl chambers and allowed the remaining N6 and BSA to pass. (Since 5ul was a big volume, it took a few minutes to empty out through the chamber outlets.)This would coat the collection channels.
- After the mix has completely passed the chambers, released all the other valves (mastermix, NS, etc.) and allowed the air to dry the channels.
- Since one air source would not completely dry the channels, another air source was placed at the TE buffer inlet at the top.
- It took about 2 hours to completely dry the channels.
- Detached the device from the manifolds and UVed the device in the crosslinker for 15mins.
- Reattached the device to the manifolds and sealed the chamber outlets with tape.
CoRE MasterMix Preparation (total 20.168ul)
- Prepare the mastermix after the cells were ready to load
- 2.33ul Phi29 10x buffer
- 8.45ul 200uM N6 primer
- 1.688ul 25mM dNTP
- 2.33ul 8% Tween20
- 2.11ul 1mM dUTP
- 0.84ul Phi29 (1ug/ul)
- 2.42ul water
- Also prepare ALS and NS
- ALS recipe: 69.5ul water + 10ul DTT(1M) + 8ul KOH(5M) + 12.5ul 8% Tween20
- NS recipe: 87.5ul NS stock + 12.5ul 8% Tween20 where NS stock: 4ml 1M HCl and 6ml 1M TrisHCl
- Preparing "negative" control: added 0.35ul of water into 10ul CoRE mastermix in PCR tube. 15 hours at 30C.
- Remaining mastermix volume was used to load the MDA device
- All reagents were loaded onto the device as in the diagram below.
MDA on Device Procedure
- Diluted cells were loaded and pushed into the cell channel (~1.5psi). I followed a single cell from the inlet and then captured.