EricChu:MDA/CoRE fragmentation protocol for microfluidic device
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MDA Device v7 Preparation[edit]
- 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)[edit]
- Prepare the mastermix after the cells were ready to load
- 2.33ul Phi29 10x buffer (from epicentre)
- 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[edit]
- Diluted cells were loaded and pushed into the cell channel (~1.5psi). I followed a single cell from the inlet and then captured next to the mixing ring.
- ALS pushed the cell into the mixing ring from the perpendicular channel.
- When the mixing ring was filled, adjusted the ALS pressure to ~2.5psi.
- 3 mins mixing in the pump
- In the meantime, NS was loaded to the device.
- Contents in the mixing ring was pushed by ALS into the ALS+DNA compartments.
- Released the valves between ALS+DNA and NS for 1-2mins.
- In the meantime, MDA mastermix was loaded to the device.
- After MDA mastermix was loaded, the ALS+DNA+NS contents were pushed into the 23nl chambers. Increased the mastermix pressure to 5psi to fill the chambers. It took about 5mins. Reduced the pressure to 2.5psi before closing all the valves for the chambers.
- NS chambers and MDA mastermix chambers were cleared by air. After 5mins, the channels were still wet.
- Moved the device to the TE block for 10-15 hours 30C and then lowered to 20C.
MDA Samples Collection from MDA Device v7[edit]
- Samples were collected by TE buffer after 1.5 hour at 20C. TE buffer: 10mM Tris, 1mM EDTA, 0.2% Tween20
- TE buffer was loaded from the top. And the samples were all pushed lower and out of the chambers to avoid mixing. The center 8 chambers were collected first. And the samples of some chambers were back flowed.
- Samples were collected by pipet tips and transferred to PCR tubes.
- All chambers were collected in about 6-7ul volume.
- 65C for 15min to deactivate Phi29
qPCR Recipe and Verification of MDA[edit]
- qPCR recipe per sample
- 8.2ul water
- 0.4ul forward B2
- 0.4ul reverse B2
- 10ul KAPA SYBR Fast (2x)
- 1ul sample
- There were a total of 24 samples; standards of 1ng/ul, 100pg/ul, 10pg/ul, and 1pg/ul; no template control (1ul water); and negative control (1ul of 1:100 dilution in water).
- Note: You cannot have a positive control.
CoRE Fragmentation Protocol[edit]
- Prepared reagents dilution and kept on ice
- 3.3x exo- Klenow buffer (from epicentre): 30ul 10x buffer + 90ul water
- diluted UDG: 1ul UDG (5000U/ml) + 128ul water
- diluted endonuclease IV: 1ul endoIV (10,000U/ml) + 128ul water
- diluted exo- Klenow: 1ul exo- Klenow + 50ul water
- diluted dNTP (from epicentre): 1ul 25mM dNTP + 25ul water
- In strip PCR tubes, added
- 1ul 3.3x exo- Klenow buffer
- 1ul diluted UDG
- 1ul diluted endonuclease
- 1ul sample from MDA device
- 37C for 2 hours and 65C for 15min
- Then added to PCR tubes
- 1ul diluted exo- Klenow
- 1ul diluted dNTP
- 37C for 1 hour and 75C for 15min
Post-CoRE Library Construction and PCR Verification (accompany to Noi/NOTES/2013-5-22)[edit]
- Samples were given to Noi
- NEBNext Multiple Adaptors were used to incorporate indexes for Illumina sequencing.
- See notes on Noi's procedure in Noi/NOTES/2013-5-22