Hosuk:Tagmentation Protocol

From ZhangLabWiki
Revision as of 20:13, 23 July 2013 by >Hosuki78
Jump to navigation Jump to search

Pol I treatment

  1. Start with 1 uL DNA
  2. Add 1.5 uL ALS, 3min
  3. Put on ice and add 1.5 uL NS
  4. Pol I Master Mix (Total 20 uL)
    • Template 4 uL
    • 10x NEB buffer 2 (NEB) 1 uL
    • 10x ampligase buffer (Epicentre) 1 uL
    • dNTP (Epicentre) 0.4 uL
    • N6(unmodified) 1 uL
    • H2O 11.6 uL
    • PolI (Invitrogen) 1 uL
  5. 37 C for 1 hr min, pause
  6. Add 0.2 uL ampligase and 37C for 10min, and 65C for 10min
  7. Standard EtOH precipitate and elute in 4 uL water
Standard EtOH precipitate
  1. Prepare 2mL Tube
  2. Mix
    • 100% EtOH 50 uL
    • 3M NAoAC 2 uL
    • Glicol Blue 1 uL
    • Sample 20 uL
  3. Put in -80C for 30min
  4. Spin at 10krpm for 25min at 4C
  5. Remove supernatant
  6. Add 750 uL of 75% EtOH
  7. Spin at 10krpm for 5min at 4C
  8. Remove supernatant
  9. Add 4 uL H2O


Tagmentation

  1. Mix (Total 10 uL)
    • DNA in water 7 uL
    • Tagmentation buffer 2 uL
    • 1:50 diluted enzyme 1 uL
  2. 55C for 5 min (1 min for sea bacteria)


  • Dilution of Tagmentation Enzyem
    • Glycerol 25 uL
    • 1x T.E. buffer 24 uL
    • Enzyme 1 uL


Protease treatment

  1. Add 1 uL 1:100 diluted protease (Qiagen)
  2. 10 min 50C, 20 min 70C


Klenew

  1. Add 1 uL exo- Klenow and .4 uL dNTP to each
  2. 37C for 15 min, 75 for 20 min


1st PCR round

  1. Mix (Total 30 uL)
    • DNA 12.4 uL
    • KAPA Fast 15 uL
    • Adapter 1 0.6 uL
    • Barcode 0.6 uL
    • H2O 1.4 uL
  2. 95 C 1min (95 C 10s, 62C 15s, 72 C 45s)x7


2nd PCR round

  1. Mix (Total 100 uL)
    • 1st round PCR mix 30 uL
    • KAPA FAST 35 uL
    • Primer 1 2 uL
    • Primer 2 2 uL
    • SYBR Green 1 0.8 uL
    • H2O 32 uL
  2. 1 min 95C, (95 C 10s, 60C 15s, 72 C 45s)x10 , 72C 3min


Bead purfication

  • Aliquot beads into smaller tube to see if it results in a better yield
  1. Add 100 uL beads to each, mix, and let sit at RT for 8min
  2. Transfer tubes to magnetic rack and let sit for 5 min
  3. Remove supernatant
  4. 2x 80% EtOH wash
  5. Sit for 15 min
  6. Elute in 20 uL water