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

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


'''Table of Components for the IVT reaction'''
'''Table of Components for the IVT reaction'''
{|  border="1" style="border-collapse:collapse;"
! Components !! No DNA Ctrl. (μl) !! GFP Ctrl. (μl) !! Target Protein (μl) with MV2 !! Target Protein (μl) with pT-CHis
|-
|format modifier (not displayed) | Hela Lysate ||12.5 || 12.5 || 12.5 || 12.5
|-
|format  |Accessory Proteins ||format | 2.5 || 2.5 || 2.5 || 2.5
|-
|format  | Reaction Mix ||format | 5 || 5 || 5 || 5
|-
|format  |pCFE-GFP DNA (0.5μg/μL) ||format | --- || 2 || --- || ---
|-
|format  | Cloned DNA (0.5 μg/μL) ||format | --- || --- || 5 || 3.3
|-
|format | Nuclease free Water ||format| 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''':
<div class="floatright">[[Image:RFP-In_vitro-PcTF_Expression.png‎ ]]<br>Human in-vitro Protein Expression
</div>
{|  border="1" style="border-collapse:collapse;" class="wikitable"
|-
! 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
|}

Revision as of 20:30, 20 December 2012

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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:


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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