EricChu:LabNotesMDA/2013-5-20: Difference between revisions
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* Remaining mastermix volume was used to load the MDA device | * Remaining mastermix volume was used to load the MDA device | ||
* All reagents were loaded onto the device as in the diagram below. | * All reagents were loaded onto the device as in the diagram below. | ||
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 | |||
[[File:MDAdeviceV7.jpg]] | [[File:MDAdeviceV7.jpg]] |
Revision as of 03:28, 23 May 2013
Use CoRE Protocol to Prepare MDA in Device v.7
- Single MEF cell was planned and used in this protocol. The difference in mastermix preparation 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.
CoRE MasterMix Preparation (total 20.168ul)
- 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
- 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.
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 File:MDAdeviceV7.jpg