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 PcTF Subcloning in E. coli
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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
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| pCFE-GFP DNA (0.5μg/μL) |
--- |
2 |
--- |
---
|
| Cloned DNA (0.5 μg/μL) |
--- |
--- |
5 |
3.3
|
| Nuclease free Water |
5 |
3 |
0 |
1.7
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- 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
Here is the result:
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PcTF Subcloning in E. coli
