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)

  1. 2.33ul Phi29 10x buffer
  2. 8.45ul 200uM N6 primer
  3. 1.688ul 25mM dNTP
  4. 2.33ul 8% Tween20
  5. 2.11ul 1mM dUTP
  6. 0.84ul Phi29 (1ug/ul)
  7. 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