Daniel:Protocols/EmulsionPCR
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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
- 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
- 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
- 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
- Optimizing experiments ran from 0.002 to 2 pmol/mL