User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/12/20: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">PcTF Subcloning in E. coli</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">PcTF Subcloning in E. coli</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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'''1-Step Human Coupled in vitro protein expression''' | '''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. | |||
[http://www.piercenet.com/browse.cfm?fldID=0F8AB1C8-B7C2-3CC6-6B59-9BC58F0BA912 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''' | |||
{| 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 | |||
|} |
Latest revision as of 22:20, 26 September 2017
PcTF Subcloning in E. coli | Main project page Previous entry Next entry | |||||||||||||||||||||||||||||||||||||||||||||||
12/20/20121-Step Human Coupled in vitro protein expression
Table of Components for the IVT reaction
Fluorescent plate reader
RFP result:
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