User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/12/20

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1-Step Human Coupled in vitro protein expression

  • 1-Step Human In Vitro Protein Expression Kits enable the translation and post-transcriptional modification of full-length proteins from mRNA or plasmid templates with yields of up to 100µg/mL per reaction.

Kit Specifications

  • The PcTF gene with two different backbones were used for the protein expression.
    • pCFE1-CHis Expression vector Concentration: 196 (ng/μL)
    • Mv2 vector , Part:BBa_J176122 Concentration: 297 (ng/μL)

Table of Components for the IVT reaction

Components No DNA Ctrl. (μl) GFP Ctrl. (μl) Target Protein (μl) with MV2 Target Protein (μl) with pT-CHis
Hela Lysate 12.5 12.5 12.5 12.5
Accessory Proteins 2.5 2.5 2.5 2.5
Reaction Mix 5 5 5 5
pCFE-GFP DNA (0.5μg/μL) --- 2 --- ---
Cloned DNA (0.5 μg/μL) --- --- 5 3.3
Nuclease free Water 5 3 0 1.7
  • The total volume of each tube should be 25 μL.
  • Incubate the reaction for 6 hrs at 30°C
  • After the incubation increase the volume of each tube up to 100 μL with Nuclease free water.

Fluorescent plate reader

  • Transfer 50 μl of each solution and a control (No DNA sample) to the Costar 96 clear bottom black side.
  • Here is the software setting:
    • Fluorescence
    • Endpoint
    • Full Plate
    • Excitation: 540, Emission: 600
    • Optics: Top
    • Gain: AutoScale
    • Light Source: Xenon Flash, Lamp Energy: High
    • Read Speed: Normal, Delay: 100 msec, Measurements/Data Point: 10
    • Read Height: 9.5 mm
  • Click OK and then Run the experiment.
  • Export the data to an excel file.
  • The software will deduce the control data from the sample data

RFP result:

RFP-In vitro-PcTF Expression.png
Human in-vitro Protein Expression

Reaction RFP (AU)
No DNA Negative Control 109
GFP Positive Control 91
Target Protein pC-CHis (Frozen) 3395
Target Protein pC-CHis (Fresh) 8819
Target Protein Mv2 0