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==
[[Media:Emulsion_PCR-Ion_Torrent.pdf|Ion Torrent Protocol (2011)]]
 
==Beads and Materials==


<ol>
<ol>
<li>Emulsions</li>
<li>ThermoFisher DynaBeads</li>
<ol type="A">
<ol type="A">
<li>Supplied as 10 mg/mL (10 ug/uL; 10<sup>10</sup> beads/mL)</li>
<li>Beads bind ~500 pmol biotin-ssDNA complex per mg</li>
<li>Church protocol (below) recommends ~10 ug of beads, which amounts to 5 pmol template (bound)</li>
</ol></ol>
==Reagents & Base Protocols==
===Emulsions===
<ol>
<li>Church Lab Protocol</li>
<li>Church Lab Protocol</li>
<ol type="a">
<ol type="a">
Line 28: Line 41:
<li>0.4% Tween 80</li>
<li>0.4% Tween 80</li>
<li>0.05% Triton X-100</li>
<li>0.05% Triton X-100</li>
<li>(Light?) Mineral oil is the organic phase solvent</li>
<li>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 [https://us.vwr.com/store/catalog/product.jsp?catalog_number=58948-377 magnetic microstir bar]</li>
<li>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 [https://us.vwr.com/store/catalog/product.jsp?catalog_number=58948-377 magnetic microstir bar]</li>
</ol>
</ol>
<li>Murgha et al</li>
<li>Murgha et al</li>
<ol type="a">
<ol type="A">
<li>4% [http://glenncorp.com/shop/abil-09/ ABIL WE 09]</li>
<li>4% [http://glenncorp.com/shop/abil-em-90/ ABIL EM 90]</li>
<li>0.05% TritonX-100</li>
<li>0.05% TritonX-100</li>
<li>Mineral oil (solvent)</li>
<li>Mineral oil (solvent)</li>
<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>
</ol></ol>
</ol>
<li>PCR protocols (Note, bolded steps are PCR repeat cycles)</li>
<li>Ion Torrent Protocol</li>
<ol type="A">
<ol type="A">
</ol>
</ol>
===PCR protocols (Note, bolded steps are PCR repeat cycles)===
<ol>
<li>Church Lab Protocol</li>
<li>Church Lab Protocol</li>
<ol type="a">
<ol type="A">
<li>96 uL 10X PCR buffer</li>
<li>96 uL 10X PCR buffer</li>
<li>360 uL 50 mM MgCl2</li>
<li>360 uL 50 mM MgCl2</li>
Line 52: Line 72:
</ol>
</ol>
<li>Shao et al Protocol</li>
<li>Shao et al Protocol</li>
<ol type="a">
<ol type="A">
<li>0.4 umol/L each primer</li>
<li>0.4 umol/L each primer</li>
<li>3.5 mmol/L MgCl2</li>
<li>3.5 mmol/L MgCl2</li>
Line 62: Line 82:
</ol>
</ol>
<li>Murgha et al</li>
<li>Murgha et al</li>
<ol type="a">
<ol type="A">
<li>2.5 fmol template</li>
<li>2.5 fmol template</li>
<li>0.5 uM each primer</li>
<li>0.5 uM each primer</li>
Line 70: Line 90:
<li>nfH2O to 100 uL</li>
<li>nfH2O to 100 uL</li>
<li>2 min 98C; '''15 sec 98 C'''; '''25 sec (Tm-2) C'''; '''25 sec 72C'''; '''goto x30'''; 5 min 72 C</li>
<li>2 min 98C; '''15 sec 98 C'''; '''25 sec (Tm-2) C'''; '''25 sec 72C'''; '''goto x30'''; 5 min 72 C</li>
</ol>
<li>Ion Torrent Protocol</li>
<ol type="A">
<li>Use the following components</li>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#6666FF;font-size:12pt;font-weight:bold" align="center"
| width="200" height="30" | Component
| width="100" | Volume
| width="110" | Final Concentration
|- style="font-size:12pt"
| height="15"  valign="bottom" | 10X PCR Buffer
| align="center" align="center" valign="bottom" | 15
| align="center" valign="bottom" | 1X
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | 25mM dNTPs
| align="center" align="center" valign="bottom" | 15
| align="center" valign="bottom" | 2.5 mM
|- style="font-size:12pt"
| height="15"  valign="bottom" | 1 mM MgCl2
| align="center" align="center" valign="bottom" | 0.5
| align="center" valign="bottom" | 3.5 mM
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | 100 uM PrimerA
| align="center" align="center" valign="bottom" | 13.5
| align="center" valign="bottom" | 9 uM
|- style="font-size:12pt"
| height="15"  valign="bottom" | 10 uM Primer B
| align="center" align="center" valign="bottom" | 1.5
| align="center" valign="bottom" | 0.1 uM
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | SNAPPS (hydrogel beads, 2M/uL)
| align="center" align="center" valign="bottom" | 10
| align="center" valign="bottom" | 50 Million
|- style="font-size:12pt"
| height="15"  valign="bottom" | CAC Polymerase (5 U/uL)
| align="center" align="center" valign="bottom" | 18
| align="center" valign="bottom" | 90 Units
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | DNA template 2nd Dilution
| align="center" align="center" valign="bottom" | 10
| align="center" valign="bottom" | 0.12 pM
|- style="font-size:12pt"
| height="15"  valign="bottom" | 1 M Ammonium Sulfate
| align="center" align="center" valign="bottom" | 0.75
| align="center" valign="bottom" | 5 mM
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | 1 mM Spermidine
| align="center" align="center" valign="bottom" | 15
| align="center" valign="bottom" | 100 uM
|- style="font-size:12pt"
| height="15"  valign="bottom" | TIPP
| align="center" align="center" valign="bottom" | 0.3
| align="center" valign="bottom" | NA
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Water
| align="center" align="center" valign="bottom" | 64.7
| align="center" valign="bottom" | NA
|- style="font-size:12pt"
| height="15"  valign="bottom" | Total Volume
|style="font-weight:bold" align="center" align="center" valign="bottom" | 150
| align="center" align="center" valign="bottom" | &nbsp;
|}
</ol>
</ol>
</ol>
</ol>
<li>Emulsion breaking</li>
 
===Emulsion breaking===
 
 
<ol>
<li>Church Lab Protocol</li>
<ol type="A">
<ol type="A">
<li>Church Lab Protocol</li>
<ol type="a">
<li>100 uL isopropanol to each well; pipette up and down >=10 times</li>
<li>100 uL isopropanol to each well; pipette up and down >=10 times</li>
<li>Vortex for 1 min; Spin down for 30 sec at 4000rpm; Remove supernatant by decanting</li>
<li>Vortex for 1 min; Spin down for 30 sec at 4000rpm; Remove supernatant by decanting</li>
Line 88: Line 189:
</ol>
</ol>
<li>Shao et al Protocol</li>
<li>Shao et al Protocol</li>
<ol type="a">
<ol type="A">
<li>Pool samples and spin at 9000g for 5 min leaving concentrated aqueous emulsion at bottom; remove supernatant</li>
<li>Pool samples and spin at 9000g for 5 min leaving concentrated aqueous emulsion at bottom; remove supernatant</li>
<li>Add 2 volumes of water-saturated ether to one volume emulsion</li>
<li>Add 2 volumes of water-saturated ether to one volume emulsion</li>
Line 95: Line 196:
</ol>
</ol>
<li>Murgha et al Protocol</li>
<li>Murgha et al Protocol</li>
<ol type="a">
<ol type="A">
<li>Successive washes with 1 mL water saturated dietyl ether and ethyl acetate (fume hood)</li>
<li>Successive washes with 1 mL water saturated dietyl ether and ethyl acetate (fume hood)</li>
<li>Final wash with diethyl ether only</li>
<li>Final wash with diethyl ether only</li>
<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>


</ol>
 
===Meltoff===
 
 
<ol>
<li>Ion Torrent</li>
<ol type="A">
<li>Resuspend the pellet in the 1.5mL tube with 100μL of TE-Triton 0.1% (TE-T)</li>
<li>Transfer the sample in the 1.5mL tube to a new 200μL PCR tube</li>
<li>Vortex the sample for 30 seconds and spin down at 13,000rpm for 1minute</li>
<li>Remove all but 20μL and add 80μL of TE-T</li>
<li>Melt off the complementary DNA strand by incubating the sample at 95°C for 3 minutes</li>
<li>Spin down sample at 13,000rpm for 1minute</li>
<li>Remove all but 20μL and keep for gel analysis</li>
<li>Add 80μL of TE-T and mix</li>
<li>Repeat steps 6 to 8 once more</li>
</ol></ol>
 
==Reagent Concentration Comparison Table==
 
 
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#9BBB59;font-size:12pt;font-weight:bold"
| align="center" width="95" height="31"  valign="bottom" | &nbsp;
| width="95" align="center" | Template
| width="95" align="center" | Primers
| width="95" align="center" | dNTP
| width="140" align="center" | Taq
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Church
| align="center" valign="bottom" | ????
| align="center" valign="bottom" | 12.5 uM
| align="center" valign="bottom" | 3.5 mM
| align="center" valign="bottom" | 0.28 U/uL
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Shao
| align="center" valign="bottom" | 10 pM
| align="center" valign="bottom" | 0.4 uM
| align="center" valign="bottom" | 0.4 mM
| align="center" valign="bottom" | 0.125E-6 U/uL
 
|- style="font-size:12pt"
| height="15"  valign="bottom" | Murgha
| align="center" valign="bottom" | 25 pM
| align="center" valign="bottom" | 0.5 uM
| align="center" valign="bottom" | 0.2 mM
| align="center" valign="bottom" | 0.04 U/uL
 
|- style="background-color:#BFBFBF;font-size:12pt"
| height="15"  valign="bottom" | Ion Torrent
| align="center" valign="bottom" | 0.12 pM
| align="center" valign="bottom" | 9 uM
| align="center" valign="bottom" | 2.5 mM
| align="center" valign="bottom" | 0.6 U/uL
 
|}
 
[[Image:SOLiD_emPCR_aqueous_phase.png|350x250px]]


==Protocol==
==Protocol==
Line 109: Line 287:
<li></li>
<li></li>
</ol>
</ol>
==Additives and Effects==
[[Image:QPCR_Additives-Roche.png|919x476px]]

Latest revision as of 20:26, 19 June 2017

Emulsion PCR[edit]

Back to Main

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)

Ion Torrent Protocol (2011)

Beads and Materials[edit]

  1. ThermoFisher DynaBeads
    1. Supplied as 10 mg/mL (10 ug/uL; 1010 beads/mL)
    2. Beads bind ~500 pmol biotin-ssDNA complex per mg
    3. Church protocol (below) recommends ~10 ug of beads, which amounts to 5 pmol template (bound)

Reagents & Base Protocols[edit]

Emulsions[edit]

  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. (Light?) Mineral oil is the organic phase solvent
    5. 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 EM 90
    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
  4. Ion Torrent Protocol

PCR protocols (Note, bolded steps are PCR repeat cycles)[edit]

  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
  4. Ion Torrent Protocol
    1. Use the following components
    2. Component Volume Final Concentration
      10X PCR Buffer 15 1X
      25mM dNTPs 15 2.5 mM
      1 mM MgCl2 0.5 3.5 mM
      100 uM PrimerA 13.5 9 uM
      10 uM Primer B 1.5 0.1 uM
      SNAPPS (hydrogel beads, 2M/uL) 10 50 Million
      CAC Polymerase (5 U/uL) 18 90 Units
      DNA template 2nd Dilution 10 0.12 pM
      1 M Ammonium Sulfate 0.75 5 mM
      1 mM Spermidine 15 100 uM
      TIPP 0.3 NA
      Water 64.7 NA
      Total Volume 150  

Emulsion breaking[edit]

  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
  4. Ion Torrent Protocol
    1. With a 200 uL pipette pool the emulsion into a 1.5 mL tube; save the tip and put aside
    2. With a new 200 uL tip add 50 uL of butanol to each PCR tube
    3. Using same tip as for pooling, clean out PCR tubes of residual emulion and add to 1.5 mL
    4. Vortex the pooled sample in the 1.5 mL tube for 30 seconds
    5. Spin down the sample 13,000 rpm for 5 minutes
    6. Take off oil-butanol mix (top layer) but do not disrupt the oil/aqueous interface
    7. Save 10 uL aqueous (bottom layer) for gel analysis, leave the rest
    8. Add 1000 uL butanol and vortex for 30 seconds
    9. Spin down sample at 13,000rpm for 3 minutes
    10. Again remove butanol without disturbing the pellet
    11. Add 500 uL 4X SSC wash buffer to 1.5 mL tube and vortex for 30 seconds
    12. Go back and mix thoroughly by pipetting up and down; the pellet may be hard to resuspend
    13. Sonicate 1 minute
    14. Spin down sample at 13,000rpm for 1 minute
    15. Remove all but 40uL wash buffer
    16. Repeat steps 11 to 15 two times (k to o)


Meltoff[edit]

  1. Ion Torrent
    1. Resuspend the pellet in the 1.5mL tube with 100μL of TE-Triton 0.1% (TE-T)
    2. Transfer the sample in the 1.5mL tube to a new 200μL PCR tube
    3. Vortex the sample for 30 seconds and spin down at 13,000rpm for 1minute
    4. Remove all but 20μL and add 80μL of TE-T
    5. Melt off the complementary DNA strand by incubating the sample at 95°C for 3 minutes
    6. Spin down sample at 13,000rpm for 1minute
    7. Remove all but 20μL and keep for gel analysis
    8. Add 80μL of TE-T and mix
    9. Repeat steps 6 to 8 once more

Reagent Concentration Comparison Table[edit]

  Template Primers dNTP Taq
Church ???? 12.5 uM 3.5 mM 0.28 U/uL
Shao 10 pM 0.4 uM 0.4 mM 0.125E-6 U/uL
Murgha 25 pM 0.5 uM 0.2 mM 0.04 U/uL
Ion Torrent 0.12 pM 9 uM 2.5 mM 0.6 U/uL

File:SOLiD emPCR aqueous phase.png

Protocol[edit]

Additives and Effects[edit]

File:QPCR Additives-Roche.png