EricChu:LabNotesMDA/2014-1-21: Difference between revisions
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>Ericchu (Created page with "==Running Real-time MDA in PCR tubes to test different MDA conditions == * Use both PGP1 cells and genomic human DNA # 1ul 400mM ALS was added to 100 cells in 1ul PBS. Incubat...") |
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* Use both PGP1 cells and genomic human DNA | * Use both PGP1 cells and genomic human DNA | ||
# 1ul 400mM ALS was added to 100 cells in 1ul PBS. Incubated for 1min in room temp. | # 1ul 400mM ALS was added to 100 cells in 1ul PBS. Incubated for 1min in room temp. | ||
# | # 1ul 400mM NS was added. Incubated for 1min in room temp. | ||
# Added 1ul of (i) water (no protease) or (ii) 24mM protease and incubate at 40C or 50C for 15min | |||
# Added 1ul of 250mM KOH (final 50mM) or 100mM KOH (final 20mM) | |||
# Added 5ul of 50mM HCl or 20mM HCl (according to the previous step). Also added 5.5ul HCl (10% extra) and 6ul HCl (20% extra) to the test. | |||
# Took 1ul out of the 10ul from above (equivalent to 10 cells) and added to 40ul MDA mix with SYBR Green. | |||
==Summary of Different Conditions== | |||
# DNA: 10 cells or 60pg human genomic DNA | |||
# Cell lysis and DNA denaturation were identical in all cases. | |||
# Neutralization: identical in all cases. | |||
# Protease: (i) absent (ii) 40C 15min or (iii) 50C 15min | |||
# DNA denaturation: final 50mM KOH or final 20mM KOH | |||
# Neutralization: (i) HCl equal molar to KOH, (ii) 10% more HCl or (iii) 20% more HCl | |||
# MDA: all identical conditions | |||
==Recipes== | |||
===CoRE Mastermix=== | |||
# 4ul 10x buffer | |||
# 4ul 8% Tween20 | |||
# 10ul N6 primer | |||
# 2ul dNTP | |||
# 2.5ul dUTP (1mM) | |||
# 4ul 10x SYBR Green | |||
# 0.4ul Phi29 (high) | |||
# 13.1ul water |
Revision as of 23:55, 29 January 2014
Running Real-time MDA in PCR tubes to test different MDA conditions
- Use both PGP1 cells and genomic human DNA
- 1ul 400mM ALS was added to 100 cells in 1ul PBS. Incubated for 1min in room temp.
- 1ul 400mM NS was added. Incubated for 1min in room temp.
- Added 1ul of (i) water (no protease) or (ii) 24mM protease and incubate at 40C or 50C for 15min
- Added 1ul of 250mM KOH (final 50mM) or 100mM KOH (final 20mM)
- Added 5ul of 50mM HCl or 20mM HCl (according to the previous step). Also added 5.5ul HCl (10% extra) and 6ul HCl (20% extra) to the test.
- Took 1ul out of the 10ul from above (equivalent to 10 cells) and added to 40ul MDA mix with SYBR Green.
Summary of Different Conditions
- DNA: 10 cells or 60pg human genomic DNA
- Cell lysis and DNA denaturation were identical in all cases.
- Neutralization: identical in all cases.
- Protease: (i) absent (ii) 40C 15min or (iii) 50C 15min
- DNA denaturation: final 50mM KOH or final 20mM KOH
- Neutralization: (i) HCl equal molar to KOH, (ii) 10% more HCl or (iii) 20% more HCl
- MDA: all identical conditions
Recipes
CoRE Mastermix
- 4ul 10x buffer
- 4ul 8% Tween20
- 10ul N6 primer
- 2ul dNTP
- 2.5ul dUTP (1mM)
- 4ul 10x SYBR Green
- 0.4ul Phi29 (high)
- 13.1ul water