Hosuk:MDA Master mix
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MDA Master Mix for CoRE[edit]
- Follow Eric's protocol
Phi29 10x buffer | 2.33 uL | |
200uM N6 primer | 8.45 uL | |
25mM dNTP | 1.688 uL | |
8% Tween20 | 2.33 uL | |
1mM dUTP | 2.11 uL | |
Phi29 (1ug/ul, High Conc. Epicentre) | 0.84 uL | |
H2O | 2.42 uL |
ALS, NS buffer[edit]
ALS
ALS, 357mM (400mM) | ALS, 133mM (149mM) | ALS, 240mM (269mM) | ||||
H2O | 69.5 uL | 74.52 uL | 72.12 uL | |||
1M DTT | 10 uL | 10 uL | 10 uL | |||
5M KOH | 8 uL | 2.98 uL | 5.38 uL | |||
8% Tween20 | 12.5 uL | 12.5 uL | 12.5 uL |
NS
NS, 357mM (400mM) | NS, 133mM (149mM) | NS, 240mM (269mM) | ||||
H2O | 0 uL | 54.9 uL | 28.66 uL | |||
NS stock | 87.5 uL | 32.6 uL | 58.84 uL | |||
8% Tween20 | 12.5 uL | 12.5 uL | 12.5 uL |
- NS stock : 4mL of 1M HCl + 6mL of 1M TrisHCl
CoRE Fragmentation protocol[edit]
- Follow Eric's protocol
- 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