Team:Cadets2Vets/Experiments

Experiments

Ticketed Cell Free Expression System Protocol

  1. 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.

  2. Work Flow

    • Identify trigger sequence and target plasmid or probe

    • Add target

    • Incubation

    • Evaluation

  3. 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

qPCR- Quantitative Polymerase Chain Reaction Protocol

  1. 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.

  2. Work Flow

    • Initial Prepartaion

    • Create master mix

    • Aliquot master mix

    • Add target DNA and standards to appropriate wells

    • Start thermocycler

  3. 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