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    Wild OpenPCR and Polymorphism Workshop
    • Notes
    • Metadata
    • Relevant Items
    • Sequential Double Digest
    • In vitro transcription of guide RNAs
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    Wild OpenPCR and Polymorphism Workshop

    Tuesday, 2/14/2017General infoHardware Construction of wild OpenPCR deviceGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5811.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5808.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5829.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5832.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5846.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5852.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5858.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5879.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5886.jpgRe-programming of Arduino Wednesday, 2/15/2017Goals of PCR test runsPCR master mix Table1PCR ProgrammGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5903.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5901.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5902.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5906.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5907.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5909.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5912.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5917.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5919.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5920.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5921.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5923.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5927.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5929.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5941.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5942.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5943.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5951.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5953.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5954.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5955.jpggel-loading-hand-notes.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5959.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5961.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5964.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5966.jpgGaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5967.jpgResultsGel-annotated-2500px.jpgConclusions & Discussions
    Tuesday, 2/14/2017
    General info
    ●
    http://openpcr.org/
    ●
    http://wlu18www30.webland.ch/wiki/Wild_OpenPCR
    ●
    https://github.com/cathalgarvey/OpenPyCR
    Hardware Construction of wild OpenPCR device
    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5811.jpg
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    obtain Wild OpenPCR PCB from Urs Gaudenz (pre-populated with SMD soldered components: analog-digital converter for the temperature sensor & chip to drive the peltier element (thanks!)
    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5808.jpg
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    start with heated lid | solder cables to lid
    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5829.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5832.jpg
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    solder PCB components
    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5846.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5852.jpg
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    connect display with ribbon cable connector
    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5858.jpg
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    drill holes and screw threads into CPU cooling device
    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5879.jpg
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    add thermal paste on both sides of peltier element
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    screw aluminium heating block to CPU cooler
    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5886.jpg
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    Re-programming of Arduino
    The OpenPCR requires that we communicate with the arduino with the ISP (Sparkfun how-to)
    The hexfile uploaded onto the ATmega16U2 turns the arduino to be recognized as a "USB key" by your computer.
    The openpcr.ino (see github, also link above) is uploaded onto the ATmega328.
    Wednesday, 2/15/2017
    Goals of PCR test runs
    ●
    check activity of different ready-to-use Taq mastermixes and compare the newly constructed PCR machines
    ○
    TaQ gene&cells (5x mix)
    ○
    ODIN (5x mix)
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    KAPA genotyping mix (2x)
    ●
    060517_pPROBE egfp-rev TGTGCCCATTAACATCACCATCTAAT 55°C (from Sachiko)
    PCR master mix
    ●
    50ul PCR reaction volume
    ●
    DNA template (Plasmid): 56ng/ul
    PCR Programm
    ●
    95°C 10min (hot starts)
    ○
    35 cycles (Stefan only 30 cycles to catch up :-))
    ●
    95°C 45sec
    ●
    55°C 30sec
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    72°C 60sec
    ●
    72°C 5min
    ●
    10°C
    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5903.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5901.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5902.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5906.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5907.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5909.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5912.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5917.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5919.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5920.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5921.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5941.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5951.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5953.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5954.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5955.jpg
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    gel-loading-hand-notes.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5959.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5961.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5964.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5966.jpg
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    GaudiLabs Wild OpenPCR and Polymorphism Workshop_Stefan_Deuber__L7A5967.jpg
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    Results
    Legend
    R1: PCR reaction mix with TaQ gene&cells (5x mix)
    R2: PCR reaction mix with ODIN (5x mix)
    R3: PCR reaction mix with KAPA genotyping mix (2x)
    PCR XYZ: PCR machine built by NAME
    negative controls: PCR reactions mixes lacking the DNA template
    Gel-annotated-2500px.jpg
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    Conclusions & Discussions
    ●
    all 3 Taq PCR master mixes (TaQ gene&cells, ODIN and KAPA) are active
    ●
    positive reactions show a band size of expected (around) 700bp
    ●
    ODIN master mix took around 1 week (at ambient temperature) for delivery (and is still active!)
    ●
    no significant difference between the 3 PCR mixes can be seen (at 35 cycles)
    ●
    all successful PCR reactions were obtained with PCR machines using standard/classical aluminum heating block with drilled holes for PCR tubes
    ●
    PCR machine "Sachiko" is using a 3D-printed steel heat block
    ●
    PCR machine "Sachiko" was struggling with Arduino code and was stuck during cycling (the absence of bands was almost expected)
    ○
    we are very curious and interested to know whether the 3D-printed steel heat block is working!

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