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 Coupled in vitro protein expression'''
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# List items
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* 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.
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[http://www.piercenet.com/browse.cfm?fldID=0F8AB1C8-B7C2-3CC6-6B59-9BC58F0BA912 Kit Specifications]
[http://www.piercenet.com/browse.cfm?fldID=0F8AB1C8-B7C2-3CC6-6B59-9BC58F0BA912 Kit Specifications]
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* The PcTF gene with two different backbones were used for the protein expression.
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** pCFE1-CHis Expression vector Concentration: 196 (ng/μL)
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** Mv2 vector , Part:BBa_J176122 Concentration: 297 (ng/μL)
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'''Table of Components for the IVT reaction'''
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{|  border="1" style="border-collapse:collapse;"
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! Components !! No DNA Ctrl. (μl) !! GFP Ctrl. (μl) !! Target Protein (μl) with MV2 !! Target Protein (μl) with pT-CHis
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|-
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|format modifier (not displayed) | Hela Lysate ||12.5 || 12.5 || 12.5 || 12.5
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|-
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|format  |Accessory Proteins ||format | 2.5 || 2.5 || 2.5 || 2.5
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|-
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|format  | Reaction Mix ||format | 5 || 5 || 5 || 5
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|-
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|format  |pCFE-GFP DNA (0.5μg/μL) ||format | --- || 2 || --- || ---
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|-
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|format  | Cloned DNA (0.5 μg/μL) ||format | --- || --- || 5 || 3.3
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|-
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|format | Nuclease free Water ||format| 5 || 3 || 0 || 1.7
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|}
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*The total volume of each tube should be 25 μL.
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*Incubate the reaction for 6 hrs at 30°C
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* After the incubation increase the volume of each tube up to 100 μL with Nuclease free water.
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'''Fluorescent plate reader'''
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* Transfer 50 μl of each solution and a control (No DNA sample) to the Costar 96 clear bottom black side.
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* Here is the software setting:
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** Fluorescence
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** Endpoint
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** Full Plate
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** Excitation: 540,  Emission: 600
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** Optics: Top
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** Gain: AutoScale
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** Light Source: Xenon Flash,  Lamp Energy: High
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** Read Speed: Normal,  Delay: 100 msec,  Measurements/Data Point: 10
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** Read Height: 9.5 mm
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* Click OK and then Run the experiment.
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* Export the data to an excel file.
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* The software will deduce the control data from the sample data
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'''RFP result''':
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<div class="floatright">[[Image:RFP-In_vitro-PcTF_Expression.png‎ ]]<br>Human in-vitro Protein Expression
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</div>
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{|  border="1" style="border-collapse:collapse;" class="wikitable"
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|-
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! Reaction  !! RFP (AU)
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|-
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| No DNA Negative Control|| 109
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|-
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| GFP Positive Control || 91
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|-
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| Target Protein pC-CHis (Frozen) || 3395
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|-
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| Target Protein pC-CHis (Fresh) || 8819
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|-
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| Target Protein Mv2 || 0
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|}

Current revision

PcTF Subcloning in E. coli Main project page
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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

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:

Image: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
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