12/20/2012

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

• 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:

Human in-vitro Protein Expression