Difference between revisions of "Team:Cadets2Vets/Experiments"

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I. Overview:                          qPCR utilizes the same basis as standard polymerase chain reaction, in which a target section of DNA can be identified and amplified via an enzymatic reaction (DNA Polymerase) and two ssDNA oligos (primers). Where qPCR differs is in the addition of either dye or probe based chemistry (this protocol will be primarily deal with the former). What this dye allows for is detection of newly formed DNA through the incorporation of a small fluorescent molecule into the new double stranded DNA (dsDNA). When the relative fluorescent units (RFU) being monitored rises above the background level (established by experimental controls) it is possible to ascertain a concentration of target gene. The relationship being the more concentrated the target the earlier in the thermocycler program signal will be generated.  
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II. Work Flow
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-Initial Preparation
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-Create master mix
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-Aliquot master mix
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-Add target DNA and standards to appropriate wells
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-Start thermocycler
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III. Reagents
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-ICE
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-Promega Hotstart Polyermase + Colorless Flexi buffer
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-10 mM dNTP
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-25 mM MgCl
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-Molecular Water
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-Forward and Reverse Primers in 10 uM
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-Evagreen dye 25x
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-Agarose
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-Sybr Safe
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Revision as of 18:22, 21 June 2017

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EXPERIMENTS - CADETS2VETS

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EXPERIMENTS.



RESEARCH. PROTOCOLS.



TICKETED CELL FREE EXPRESSION SYSTEM PROTOCOLS



I. Overview

The idea of cell free expression has existed for more than 20 years. It is the notion of taking the elements required for both transcription and translation and utilizing them for an external protein expression system. This is extremely beneficial for expression of cytotoxic proteins in which high yields are difficult to produce in vivo. For our purpose within this assay, this concept is utilized in conjunction with a regulatory factor for detection. This is detection is predicated either by regulation of the RBS at the transcript level or by sequestering the binding sight of RNA polymerase.

II. Work Flow

§ Identify trigger sequence and target plasmid or probe

§ Add target

§ Incubation

§ Evaluation

III. Reagents

§ Cell free expression kit

§ •Promega L5540

§ •Promega L1020

§ •Promega L1110

§ •NEB E6800L

§ •Life Tech K990100

§ RNASE Inhibitor (RI)

§ Prepared tickets

§ Trigger Plasmid

§ Substrate if needed



aPCR- Quantitative Polymerase Chain Reaction Protocol







  • I. Overview: qPCR utilizes the same basis as standard polymerase chain reaction, in which a target section of DNA can be identified and amplified via an enzymatic reaction (DNA Polymerase) and two ssDNA oligos (primers). Where qPCR differs is in the addition of either dye or probe based chemistry (this protocol will be primarily deal with the former). What this dye allows for is detection of newly formed DNA through the incorporation of a small fluorescent molecule into the new double stranded DNA (dsDNA). When the relative fluorescent units (RFU) being monitored rises above the background level (established by experimental controls) it is possible to ascertain a concentration of target gene. The relationship being the more concentrated the target the earlier in the thermocycler program signal will be generated.

    II. Work Flow

  • -Initial Preparation

  • -Create master mix

  • -Aliquot master mix

  • -Add target DNA and standards to appropriate wells

  • -Start thermocycler

    III. Reagents

  • -ICE

  • -Promega Hotstart Polyermase + Colorless Flexi buffer

  • -10 mM dNTP

  • -25 mM MgCl

  • -Molecular Water

  • -Forward and Reverse Primers in 10 uM

  • -Evagreen dye 25x

  • -Agarose

  • -Sybr Safe



    • Experimental outline





    I. Pre-Phase One Experiments

    Establish control experiments

    § -Constitutive GFP

    § o Switch H GFP

    II. Pre-Phase One Goals

    § The goal of pre-phase 1 is to establish a working positive control and negative control in lab.

    III. Phase One Experiments

    § Clone composite circuit in e.coli

    § Test efficacy of the circuit in vivo and determine threshold limits of both arsenic and arsenite

    § Perform mini prep and quantify

    IV. Phase One Goals

    § The goals of this phase is to grow a large amount of the composite circuit and develop an understanding of whether or not the circuit is biologically effective. Another primary goal is to develop an understanding of how the circuit works in its entirety (upper threshold/lower threshold)

    V. Phase Two Experiments

    § Test the efficacy of the composite circuit instead of crude lysate

    § Test purified plasmid with Promega S30 kit

    VI. Phase Two Goals

    § The goal of this phase is to take the circuit out of the cell and test it in situ utilizing both a bacterial expression kit (Promega S30) and an attempt at functionalizing the circuit utilizing a formulated crude lysate for increased application space.

    VII. Phase Three Experiments

    § Attempt at micro scaling reaction

    § Porting reactions to paper based system

    § Attempt at lyophilizing the circuit on paper both with Promega S30 kit and in the crude lysate form (if the previous phase demonstrated reasonable evidence for progressing on that front)

    § Phase Three Goals

    § The goal of this phase is to successfully micro scale the reaction and port it to a lateral flow paper based system. If successful in this phase, we will have developed a biological means to detect presence of arsenic and arsenite utilizing a lateral flow paper system that is both cost effective and easy to implement.


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