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

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'''PCR Reactions'''
'''PCR Reactions'''


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{|   class="wikitable" border=1 cellpadding="7" cellspacing="0"
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''Part'''
| align="center" style="background:#f0f0f0;"|'''BD002'''
| align="center" style="background:#f0f0f0;"|'''BD002'''
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| Tolal (µL)||100||100||100
| Tolal (µL)||100||100||100
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Revision as of 15:23, 24 October 2013

PcTF Subcloning in E. coli <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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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
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
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