Hosuk:Tagmentation Protocol
Jump to navigation
Jump to search
Pol I treatment[edit]
- Start with 1 uL DNA --> 2uL DNA from MDA device
- Add 1.5 uL ALS, 3min --> 3uL ALS with 400mM KOH
- Put on ice and add 1.5 uL NS --> 3uL NS
- Pol I Master Mix (Total 20 uL)
- Template 4 uL --> 7 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 --> 8.6 uL
- PolI (Invitrogen) 1 uL
- 37 C for 1 hr min, pause
- Add 0.2 uL ampligase and 37C for 10min, and 65C for 10min
- Standard EtOH precipitate and elute in 4 uL water
Standard EtOH precipitate[edit]
- Prepare 2mL Tube
- Mix
- 100% EtOH 50 uL
- 3M NAoAC 2 uL
- Glicol Blue 1 uL
- Sample 20 uL
- Put in -80C for 30min
- Spin at 10krpm for 25min at 4C
- Remove supernatant
- Add 750 uL of 75% EtOH
- Spin at 10krpm for 5min at 4C
- Remove supernatant (pipette 700 uL first --> 200 uL pipette last)
- Elute with 7 uL H2O
Tagmentation[edit]
- Mix (Total 10 uL)
- DNA in water 7 uL
- Tagmentation buffer 2 uL
- 1:50 diluted enzyme 1 uL
- 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[edit]
- Add 1 uL 1:100 diluted protease (Qiagen)
- 10 min 50C, 20 min 70C
- Protease (Qiagen)
- 7.5 AU in stock
- Resuspension to 5 AU/mL
Klenew[edit]
- Add 1 uL exo- Klenow and .4 uL dNTP to each
- 37C for 15 min, 75 for 20 min
1st PCR round[edit]
- 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
- 95 C 1min (95 C 10s, 62C 15s, 72 C 45s)x7
2nd PCR round[edit]
- 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
- 1 min 95C, (95 C 10s, 60C 15s, 72 C 45s)x10 , 72C 3min
Bead purfication[edit]
- Aliquot beads into smaller tube to see if it results in a better yield
- Add 100 uL beads to each, mix, and let sit at RT for 8min
- Transfer tubes to magnetic rack and let sit for 5 min
- Remove supernatant
- 2x 80% EtOH wash
- Sit for 15 min
- Elute in 20 uL water