Daniel:Protocols/EmulsionPCR: Difference between revisions
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>Djacobse |
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(8 intermediate revisions by the same user not shown) | |||
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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)]] | ||
[[Media:Emulsion_PCR-Ion_Torrent.pdf|Ion Torrent Protocol (2011)]] | |||
==Beads and Materials== | ==Beads and Materials== | ||
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<li>Murgha et al</li> | <li>Murgha et al</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>4% [http://glenncorp.com/shop/abil- | <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> | ||
Line 218: | Line 220: | ||
<li>Repeat steps 11 to 15 two times (k to o)</li></ol> | <li>Repeat steps 11 to 15 two times (k to o)</li></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" | | |||
| 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 224: | 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]
Protocol and tips for emulsion PCR. Used for amplification of highly uniform DNA oligos on streptavidin beads (commonly).
Church lab emulsion PCR protocol (2007)
Beads and Materials[edit]
- ThermoFisher DynaBeads
- Supplied as 10 mg/mL (10 ug/uL; 1010 beads/mL)
- Beads bind ~500 pmol biotin-ssDNA complex per mg
- Church protocol (below) recommends ~10 ug of beads, which amounts to 5 pmol template (bound)
Reagents & Base Protocols[edit]
Emulsions[edit]
- 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 EM 90
- 0.05% TritonX-100
- Mineral oil (solvent)
- Add aqueous to oil and stir at 1000 rpm at 4C; stir additional 15 minutes after mixing
- Ion Torrent Protocol
PCR protocols (Note, bolded steps are PCR repeat cycles)[edit]
- 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
- Ion Torrent Protocol
- Use the following components
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]
- 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)
Meltoff[edit]
- Ion Torrent
- Resuspend the pellet in the 1.5mL tube with 100μL of TE-Triton 0.1% (TE-T)
- Transfer the sample in the 1.5mL tube to a new 200μL PCR tube
- Vortex the sample for 30 seconds and spin down at 13,000rpm for 1minute
- Remove all but 20μL and add 80μL of TE-T
- Melt off the complementary DNA strand by incubating the sample at 95°C for 3 minutes
- Spin down sample at 13,000rpm for 1minute
- Remove all but 20μL and keep for gel analysis
- Add 80μL of TE-T and mix
- 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