User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/12/20: Difference between revisions
From OpenWetWare
(3 intermediate revisions by the same user not shown) | |||
Line 14: | Line 14: | ||
* 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" | {| 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 | ! Components !! No DNA Ctrl. (μl) !! GFP Ctrl. (μl) !! Target Protein (μl) with MV2 !! Target Protein (μl) with pT-CHis | ||
|- | |- | ||
Line 32: | Line 32: | ||
|- | |- | ||
|format | Nuclease free Water ||format| 5 || 3 || 0 || 1.7 | |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
PcTF Subcloning in E. coli | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |||||||||||||||||||||||||||||||||||||||||||||||
12/20/20121-Step Human Coupled in vitro protein expression
Table of Components for the IVT reaction
Fluorescent plate reader
RFP result:
|