Hosuk:MDA Master mix

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MDA Master Mix for CoRE[edit]

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]


  • Prepared reagents dilution and kept on ice
  1. 3.3x exo- Klenow buffer (from epicentre): 30ul 10x buffer + 90ul water
  2. diluted UDG: 1ul UDG (5000U/ml) + 128ul water
  3. diluted endonuclease IV: 1ul endoIV (10,000U/ml) + 128ul water
  4. diluted exo- Klenow: 1ul exo- Klenow + 50ul water
  5. diluted dNTP (from epicentre): 1ul 25mM dNTP + 25ul water


  • In strip PCR tubes, added
  1. 1ul 3.3x exo- Klenow buffer
  2. 1ul diluted UDG
  3. 1ul diluted endonuclease
  4. 1ul sample from MDA device
  • 37C for 2 hours and 65C for 15min


  • Then added to PCR tubes
  1. 1ul diluted exo- Klenow
  2. 1ul diluted dNTP
  • 37C for 1 hour and 75C for 15min