User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/17: Difference between revisions
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| align="center" style="background:#f0f0f0;"|'''HPk''' | | align="center" style="background:#f0f0f0;"|'''HPk''' | ||
| align="center" style="background:#f0f0f0;"|'''CMV''' | | align="center" style="background:#f0f0f0;"|'''CMV''' | ||
| align="center" style="background:#f0f0f0;"|'''Reaction Conditions''' | |||
| align="center" style="background:#f0f0f0;"|'''Gel Picture''' | |||
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|{| DNA Template (µL)||0.1||0.1||0.1 || rowspan="6" | [[Image:20131020 171748 (2).jpg |thumb|300px| Column 1 is the vector backbone size: ~4600 bps, column 2 is the amplified HPK promoter size~516bps and column 3 is amplified CMV size: 588bps ]] | |{| DNA Template (µL)||0.1||0.1||0.1 || rowspan="6" | | ||
* 95°C, 3 min. | |||
* [95°C, 30 sec.; '''45'''°C, 40 sec.; 72°C, '''6''' min.] x35 | |||
* 72°C, 3 min. | |||
* 4°C, ∞ | |||
|rowspan="6" | [[Image:20131020 171748 (2).jpg |thumb|300px| Column 1 is the vector backbone size: ~4600 bps, column 2 is the amplified HPK promoter size~516bps and column 3 is amplified CMV size: 588bps ]] | |||
|- | |- | ||
| Forward Promer (µL)||1||1||1 | | Forward Promer (µL)||1||1||1 |
Revision as of 16:17, 24 October 2013
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> | ||||||||||||||||||||||||||||||||
Golden Gate Assembly of BD002 to Replace HsvtkTATA with CMV and HPK PromotersDesired Constructs: BD003: 5XGAL4-Spacer-CMV-Kozak-AMCyan-AmCyan-NLS-Stop BD004: 5XGAL4-Spacer-HPK-Kozak-AMCyan-AmCyan-NLS-Stop Existing construct is: 5XGAL4-Spacer-HsvTK-Kozak-AMCyan-AmCyan-NLS-Stop in V0200 mammalian vector Amplification of CMV and HPK from KAH187 and KAH184 respectively. The backbone vector has BSMBI cut site within the hygromycine resistance gene. I was removed though Site Directed Mutagenesis PCR Reactions
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