Daniel:Protocols/EmulsionPCR: Difference between revisions

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<li>Add aqueous to oil and stir at 1000 rpm at 4C; stir additional 15 minutes after mixing</li>
<li>Add aqueous to oil and stir at 1000 rpm at 4C; stir additional 15 minutes after mixing</li>
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</ol></ol>
<li>PCR protocols</li>
<li>PCR protocols (Note, bolded steps are PCR repeat cycles)</li>
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<ol type="A">
<li>Church Lab Protocol</li>
<li>Church Lab Protocol</li>

Revision as of 21:09, 10 April 2017

Emulsion PCR

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Protocol and tips for emulsion PCR. Used for amplification of highly uniform DNA oligos on streptavidin beads (commonly).

Shao Emulsion PCR (2011)

Church lab emulsion PCR protocol (2007)

Murgha et al protocol (2014)

Reagents

  1. Emulsions
    1. Church Lab Protocol
      1. 4.01 mL Tegosoft DEC
      2. 1.10 mL light mineral oil
      3. 385 uL ABIL WE 09
      4. Add 960 uL aqueous solution in 5.5 mL oil phase in a 50 mL falcon tube; vortex 5 minutes
    2. Shao et al Protocol
      1. 4.5% Span 80
      2. 0.4% Tween 80
      3. 0.05% Triton X-100
      4. 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
    3. Murgha et al
      1. 4% ABIL WE 09
      2. 0.05% TritonX-100
      3. Mineral oil (solvent)
      4. Add aqueous to oil and stir at 1000 rpm at 4C; stir additional 15 minutes after mixing
  2. PCR protocols (Note, bolded steps are PCR repeat cycles)
    1. Church Lab Protocol
      1. 96 uL 10X PCR buffer
      2. 360 uL 50 mM MgCl2
      3. 135 uL 25 mM (each) dNTP
      4. 6.0 uL 2 mM PR1R-S
      5. 60 uL MyOne beads in TE, pre-loaded with former primer
      6. 54 uL 5 U/uL hot-start Taq
      7. 1.0 uL template DNA (??? concentration)
      8. 248 uL nfH2O
      9. 10 min 94C; 15 sec 94 C; 30 sec 57C; 75 sec 70C; goto x119; 2 min 70 C; 4C forever
    2. Shao et al Protocol
      1. 0.4 umol/L each primer
      2. 3.5 mmol/L MgCl2
      3. 0.4 mmol/L each dNTP
      4. 0.125 unit/L Taq polymerase
      5. 0.01 pmol/mL ssDNA template
      6. nfH2O to 100 uL
      7. 2 min 94C; 30 sec 94 C; 30 sec 65C; 30 sec 72C
    3. Murgha et al
      1. 2.5 fmol template
      2. 0.5 uM each primer
      3. 0.2 mM each dNTP
      4. 0.5 ug/uL bovine serum albumin
      5. 4 Units of Phusion HS polymerase and 1x GC rich buffer
      6. nfH2O to 100 uL
      7. 2 min 98C; 15 sec 98 C; 25 sec (Tm-2) C; 25 sec 72C; goto x30; 5 min 72 C
      8. Optimizing experiments ran from 0.002 to 2 pmol/mL

Protocol