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Hosuk:Tagmentation Protocol
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===Pol I treatment=== #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===== #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=== #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=== #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=== #Add 1 uL exo- Klenow and .4 uL dNTP to each #37C for 15 min, 75 for 20 min ===1st PCR round=== #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=== #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=== *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
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