Revision as of 23:22, 20 December 2012

PcTF Subcloning in E. coli

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.

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:

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

@@ Line 18: / Line 18: @@
 '''Table of Components for the IVT reaction'''
-{| border="1"
+{|  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
 |-