EricChu:LabNotesMDA/2014-2-10
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Use of Qiagen Protease and Gibco Trypsin in Real Time MDA
- Tried Roche cOmplete protease inhibitor on both Protease and Trypsin. 1.5ml water was added to one mini tablet to make 7x inhibitor cocktail.
- Protease was diluted in water to 24mAU.
- Trypsin was diluted to 0.5x in water.
- 1000 PGP1 cells in 10ul were used. 10ul 400mM KOH denaturation buffer was added and incubated for 3min in room temperature. Then 10ul 400um HCl neutralization buffer was added and transferred to ice.
- Heat deactivating protease: 3ul of the above (~100 cell lysate) was added to 1ul protease and then transferred to 45C for 15mins and 75C for 15mins. 1ul water was added. (5ul volume)
- Inhibitor for protease: 3ul of the above (~100 cell lysate) was added to 1ul protease and then transferred to 45C for 15mins. 1ul inhibitor was added. (5ul volume)
- No inhibitor for protease: 3ul of the above (~100 cell lysate) was added to 1ul protease and then transferred to 45C for 15mins. 1ul water was then added. (5ul volume)
- 100mM KOH for protease: 3ul of the above (~100 cell lysate) was added to 1ul 400mM KOH. 1ul water was then added. (5ul volume)
- 50mM KOH for protease: 3ul of the above (~100 cell lysate) was added to 1ul 200mM KOH. 1ul water was then added. (5ul volume)
- Normal MDA: 3ul of the above was added to 2ul water.
- Inhibitor for Trypsin: 3ul of the above was added to 1ul 0.5x trypsin. 37C for 15min and then added 1ul inhibitor.
- No inhibitor for Trypsin: 3ul of the above was added to 2ul water.
- MDA master mix (40ul) was prepared as the following:
- 4ul 10x buffer
- 4ul 8% Tween20
- 10ul N6 primer
- 2ul dNTP
- 2.5ul dUTP (1mM)
- 4ul 10x SYBR Green
- 1ul Phi29 (low concentration)
- 12.5ul water
- 1ul of each of the above conditions was added to 40ul MDA master mix in the white strip PCR tubes.
- MDA was performed at 30C and measurement was taken at each 6mins.