Daniel:Protocols/EmulsionPCR: Difference between revisions

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[[Media:Murgha_Methods_For_ssOligo_Libraries_PLOS_One_2014.PDF|Murgha et al protocol (2014)]]
[[Media:Murgha_Methods_For_ssOligo_Libraries_PLOS_One_2014.PDF|Murgha et al protocol (2014)]]


===Reagents===
==Reagents==


<ol>
<ol>

Revision as of 21:29, 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
  3. Emulsion breaking
    1. Church Lab Protocol
      1. 100 uL isopropanol to each well; pipette up and down >=10 times
      2. Vortex for 1 min; Spin down for 30 sec at 4000rpm; Remove supernatant by decanting
      3. Add 5 mL isopropanol & resuspend via pipetting >1 min; Add 5 mL isopropanol; Vortex 30 sec; Spin down for 30 sec at 3000rpm
      4. Remove supernatant using magnetic particle separator
      5. Resuspend in 1 mL NXS and pipette for >1 min; transfer to 1.5 siliconized eppendorf
      6. Vortex 30 sec; spin for 30 sec at 1000g; Use magnet to remove supernatant
      7. Resuspend in 1 mL TE; mix by pipetting >30sec; spin for 30 sec at 1000g; use magnet to remove supernatant
      8. Resuspend in 500 uL TE buffer; use magnet to remove supernatant
      9. Resuspend in 500 uL 0.1M NaOH; incubate on labquake at RT for 5 min (1.5 mL mixer)
      10. Wash 1X with 500 uL 0.1M NaOH; Wash 2X with 500 uL TE; Resuspend in 30 uL TE
    2. Shao et al Protocol
      1. Pool samples and spin at 9000g for 5 min leaving concentrated aqueous emulsion at bottom; remove supernatant
      2. Add 2 volumes of water-saturated ether to one volume emulsion
      3. Vortex and centrifuge to remove ether
      4. Wash aqueous phase two times with ether and dry at room temperature
    3. Murgha et al Protocol
      1. Successive washes with 1 mL water saturated dietyl ether and ethyl acetate (fume hood)
      2. Final wash with diethyl ether only
      3. Evaporate diethyl ether by incubating 10-15 min at 37C
      4. Purify with Qiaquick column with additional washing step

Protocol