User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/17

From OpenWetWare
Jump to navigationJump to search
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>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</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 Promoters

Desired 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

Part BD002 HPk CMV
DNA Template (µL) 0.1 0.1 0.1
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
Reverse Primer (µL) 1 1 1
2X GoTag (µL) 50 50 50
dH2O (µL) 47.9 47.9 47.9
Tolal (µL) 100 100 100
Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli/2013/10/17&oldid=744460"