Daniel:Protocols/EmulsionPCR: Difference between revisions
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<li>Evaporate diethyl ether by incubating 10-15 min at 37C</li> | <li>Evaporate diethyl ether by incubating 10-15 min at 37C</li> | ||
<li>Purify with Qiaquick column '''with additional washing step'''</li></ol> | <li>Purify with Qiaquick column '''with additional washing step'''</li></ol> | ||
<li>Ion Torrent Protocol</li> | |||
<ol type="a"> | |||
<li>With a 200 uL pipette pool the emulsion into a 1.5 mL tube; save the tip and put aside</li> | |||
<li>With a new 200 uL tip add 50 uL of butanol to each PCR tube</li> | |||
<li>Using same tip as for pooling, clean out PCR tubes of residual emulion and add to 1.5 mL</li> | |||
<li>Vortex the pooled sample in the 1.5 mL tube for 30 seconds</li> | |||
<li>Spin down the sample 13,000 rpm for 5 minutes</li> | |||
<li>Take off oil-butanol mix (top layer) but do not disrupt the oil/aqueous interface</li> | |||
<li>Save 10 uL aqueous (bottom layer) for gel analysis, leave the rest</li> | |||
<li>Add 1000 uL butanol and vortex for 30 seconds</li> | |||
<li>Spin down sample at 13,000rpm for 3 minutes</li> | |||
<li>Again remove butanol without disturbing the pellet</li> | |||
<li>Add 500 uL 4X SSC wash buffer to 1.5 mL tube and vortex for 30 seconds</li> | |||
<li>Go back and mix thoroughly by pipetting up and down; the pellet may be hard to resuspend</li> | |||
<li>Sonicate 1 minute</li> | |||
<li>Spin down sample at 13,000rpm for 1 minute</li> | |||
<li>Remove all but 40uL wash buffer</li> | |||
<li>Repeat steps 11 to 15 two times (k to o)</li></ol> | |||
</ol> | </ol> | ||
Revision as of 22:51, 14 April 2017
Emulsion PCR
Protocol and tips for emulsion PCR. Used for amplification of highly uniform DNA oligos on streptavidin beads (commonly).
Church lab emulsion PCR protocol (2007)
Reagents & Base Protocols
- Emulsions
- Church Lab Protocol
- 4.01 mL Tegosoft DEC
- 1.10 mL light mineral oil
- 385 uL ABIL WE 09
- Add 960 uL aqueous solution in 5.5 mL oil phase in a 50 mL falcon tube; vortex 5 minutes
- Shao et al Protocol
- 4.5% Span 80
- 0.4% Tween 80
- 0.05% Triton X-100
- (Light?) Mineral oil is the organic phase solvent
- Mix 100 uL aqueous, 10 uL at a time, into 200 uL oil phase in a 2 mL cryo-vial continuously stirred at 1500 rpm with a magnetic microstir bar
- Murgha et al
- 4% ABIL WE 09
- 0.05% TritonX-100
- Mineral oil (solvent)
- Add aqueous to oil and stir at 1000 rpm at 4C; stir additional 15 minutes after mixing
- PCR protocols (Note, bolded steps are PCR repeat cycles)
- Church Lab Protocol
- 96 uL 10X PCR buffer
- 360 uL 50 mM MgCl2
- 135 uL 25 mM (each) dNTP
- 6.0 uL 2 mM PR1R-S
- 60 uL MyOne beads in TE, pre-loaded with former primer
- 54 uL 5 U/uL hot-start Taq
- 1.0 uL template DNA (??? concentration)
- 248 uL nfH2O
- 10 min 94C; 15 sec 94 C; 30 sec 57C; 75 sec 70C; goto x119; 2 min 70 C; 4C forever
- Shao et al Protocol
- 0.4 umol/L each primer
- 3.5 mmol/L MgCl2
- 0.4 mmol/L each dNTP
- 0.125 unit/L Taq polymerase
- 0.01 pmol/mL ssDNA template
- nfH2O to 100 uL
- 2 min 94C; 30 sec 94 C; 30 sec 65C; 30 sec 72C
- Murgha et al
- 2.5 fmol template
- 0.5 uM each primer
- 0.2 mM each dNTP
- 0.5 ug/uL bovine serum albumin
- 4 Units of Phusion HS polymerase and 1x GC rich buffer
- nfH2O to 100 uL
- 2 min 98C; 15 sec 98 C; 25 sec (Tm-2) C; 25 sec 72C; goto x30; 5 min 72 C
- Emulsion breaking
- Church Lab Protocol
- 100 uL isopropanol to each well; pipette up and down >=10 times
- Vortex for 1 min; Spin down for 30 sec at 4000rpm; Remove supernatant by decanting
- Add 5 mL isopropanol & resuspend via pipetting >1 min; Add 5 mL isopropanol; Vortex 30 sec; Spin down for 30 sec at 3000rpm
- Remove supernatant using magnetic particle separator
- Resuspend in 1 mL NXS and pipette for >1 min; transfer to 1.5 siliconized eppendorf
- Vortex 30 sec; spin for 30 sec at 1000g; Use magnet to remove supernatant
- Resuspend in 1 mL TE; mix by pipetting >30sec; spin for 30 sec at 1000g; use magnet to remove supernatant
- Resuspend in 500 uL TE buffer; use magnet to remove supernatant
- Resuspend in 500 uL 0.1M NaOH; incubate on labquake at RT for 5 min (1.5 mL mixer)
- Wash 1X with 500 uL 0.1M NaOH; Wash 2X with 500 uL TE; Resuspend in 30 uL TE
- Shao et al Protocol
- Pool samples and spin at 9000g for 5 min leaving concentrated aqueous emulsion at bottom; remove supernatant
- Add 2 volumes of water-saturated ether to one volume emulsion
- Vortex and centrifuge to remove ether
- Wash aqueous phase two times with ether and dry at room temperature
- Murgha et al Protocol
- Successive washes with 1 mL water saturated dietyl ether and ethyl acetate (fume hood)
- Final wash with diethyl ether only
- Evaporate diethyl ether by incubating 10-15 min at 37C
- Purify with Qiaquick column with additional washing step
- Ion Torrent Protocol
- With a 200 uL pipette pool the emulsion into a 1.5 mL tube; save the tip and put aside
- With a new 200 uL tip add 50 uL of butanol to each PCR tube
- Using same tip as for pooling, clean out PCR tubes of residual emulion and add to 1.5 mL
- Vortex the pooled sample in the 1.5 mL tube for 30 seconds
- Spin down the sample 13,000 rpm for 5 minutes
- Take off oil-butanol mix (top layer) but do not disrupt the oil/aqueous interface
- Save 10 uL aqueous (bottom layer) for gel analysis, leave the rest
- Add 1000 uL butanol and vortex for 30 seconds
- Spin down sample at 13,000rpm for 3 minutes
- Again remove butanol without disturbing the pellet
- Add 500 uL 4X SSC wash buffer to 1.5 mL tube and vortex for 30 seconds
- Go back and mix thoroughly by pipetting up and down; the pellet may be hard to resuspend
- Sonicate 1 minute
- Spin down sample at 13,000rpm for 1 minute
- Remove all but 40uL wash buffer
- Repeat steps 11 to 15 two times (k to o)