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=Emulsion PCR= [[Daniel Jacobsen|Back to Main]] Protocol and tips for emulsion PCR. Used for amplification of highly uniform DNA oligos on streptavidin beads (commonly). [[Media:Shao_Emulsion_PCR_Efficient_DNA_Amplification_PLOS_2011.PDF|Shao Emulsion PCR (2011)]] [[Media:Church_Lab_Emulsion_PCR_Protocol.pdf|Church lab emulsion PCR protocol (2007)]] [[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== <ol> <li>ThermoFisher DynaBeads</li> <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> <ol type="a"> <li>4.01 mL Tegosoft [http://www.sigmaaldrich.com/catalog/product/aldrich/d91551?lang=en®ion=US DEC]</li> <li>1.10 mL [http://www.sigmaaldrich.com/catalog/product/sigma/m5904?lang=en®ion=US light mineral oil]</li> <li>385 uL [http://glenncorp.com/shop/abil-09/ ABIL WE 09]</li> <li>Add 960 uL aqueous solution in 5.5 mL oil phase in a 50 mL falcon tube; vortex 5 minutes</li> </ol> <li>Shao et al Protocol</li> <ol type="a"> <li>4.5% Span 80</li> <li>0.4% Tween 80</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> </ol> <li>Murgha et al</li> <ol type="A"> <li>4% [http://glenncorp.com/shop/abil-em-90/ ABIL EM 90]</li> <li>0.05% TritonX-100</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> </ol> <li>Ion Torrent Protocol</li> <ol type="A"> </ol> </ol> ===PCR protocols (Note, bolded steps are PCR repeat cycles)=== <ol> <li>Church Lab Protocol</li> <ol type="A"> <li>96 uL 10X PCR buffer</li> <li>360 uL 50 mM MgCl2</li> <li>135 uL 25 mM (each) dNTP</li> <li>6.0 uL 2 mM PR1R-S</li> <li>60 uL MyOne beads in TE, pre-loaded with former primer</li> <li>54 uL 5 U/uL hot-start Taq</li> <li>1.0 uL template DNA (??? concentration)</li> <li>248 uL nfH2O</li> <li>10 min 94C; '''15 sec 94 C'''; '''30 sec 57C'''; '''75 sec 70C'''; '''goto x119'''; 2 min 70 C; 4C forever</li> </ol> <li>Shao et al Protocol</li> <ol type="A"> <li>0.4 umol/L each primer</li> <li>3.5 mmol/L MgCl2</li> <li>0.4 mmol/L each dNTP</li> <li>0.125 unit/L Taq polymerase</li> <li>0.01 pmol/mL ssDNA template</li> <li>nfH2O to 100 uL</li> <li>2 min 94C; '''30 sec 94 C'''; '''30 sec 65C'''; '''30 sec 72C'''</li> </ol> <li>Murgha et al</li> <ol type="A"> <li>2.5 fmol template</li> <li>0.5 uM each primer</li> <li>0.2 mM each dNTP</li> <li>0.5 ug/uL bovine serum albumin</li> <li>4 Units of Phusion HS polymerase and 1x GC rich buffer</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> </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" | |} </ol> </ol> ===Emulsion breaking=== <ol> <li>Church Lab Protocol</li> <ol type="A"> <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>Add 5 mL isopropanol & resuspend via pipetting >1 min; Add 5 mL isopropanol; Vortex 30 sec; Spin down for 30 sec at 3000rpm</li> <li>Remove supernatant using magnetic particle separator</li> <li>Resuspend in 1 mL NXS and pipette for >1 min; transfer to 1.5 siliconized eppendorf</li> <li>Vortex 30 sec; spin for 30 sec at 1000g; Use magnet to remove supernatant</li> <li>Resuspend in 1 mL TE; mix by pipetting >30sec; spin for 30 sec at 1000g; use magnet to remove supernatant</li> <li>Resuspend in 500 uL TE buffer; use magnet to remove supernatant</li> <li>Resuspend in 500 uL 0.1M NaOH; incubate on labquake at RT for 5 min (1.5 mL mixer)</li> <li>Wash 1X with 500 uL 0.1M NaOH; Wash 2X with 500 uL TE; Resuspend in 30 uL TE</li> </ol> <li>Shao et al Protocol</li> <ol type="A"> <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>Vortex and centrifuge to remove ether</li> <li>Wash aqueous phase two times with ether and dry at room temperature</li> </ol> <li>Murgha et al Protocol</li> <ol type="A"> <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>Evaporate diethyl ether by incubating 10-15 min at 37C</li> <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> ===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== <ol> <li></li> </ol> ==Additives and Effects== [[Image:QPCR_Additives-Roche.png|919x476px]]
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