Revision as of 20:14, 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
- Mv2 vector , Part:BBa_J176122

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

@@ Line 21: / Line 21: @@
 ! 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 ||43125
+|format modifier (not displayed) | Hela Lysate ||12.5 || 12.5 || 12.5 || 12.5
 |-
-|format  |Accessory Proteins ||format | 16143
+|format  |Accessory Proteins ||format | 2.5 || 2.5 || 2.5 || 2.5
 |-
-|format  | Reaction Mix ||format | 7978
+|format  | Reaction Mix ||format | 5 || 5 || 5 || 5
 |-
-|format  |pCFE-GFP DNA (0.5μg/μL) ||format | 4124
+|format  |pCFE-GFP DNA (0.5μg/μL) ||format | --- || 2 || --- || ---
 |-
-|format  | Cloned DNA (0.5 μg/μL) ||format | 2718
+|format  | Cloned DNA (0.5 μg/μL) ||format | --- || --- || 5 || 3.3
 |-
-|format | Nuclease free Water ||format|
+|format | Nuclease free Water ||format| 5 || 3 || 0 || 1.7
 |}