User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/17: Difference between revisions
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** Formula: x = length in bp ÷ measured ng/μL * 0.013 * 20 | ** Formula: x = length in bp ÷ measured ng/μL * 0.013 * 20 | ||
'''Backbone vector <math> X= (4600 / 209 ) * 0.013 * 20 = 5.72</math> | '''Backbone vector''' <math> X= (4600 / 209 ) * 0.013 * 20 = 5.72</math> | ||
'''HPK''' <math> X= (2203 / 70 ) * 0.013 * 20 = 8.18</math> | '''HPK''' <math> X= (2203 / 70 ) * 0.013 * 20 = 8.18</math> | ||
'''CMV''' <math> X= (2275 / 42 ) * 0.013 * 20 = 14.08</math> | '''CMV''' <math> X= (2275 / 42 ) * 0.013 * 20 = 14.08</math> | ||
{| class="wikitable" border=1 cellpadding="7" cellspacing="0" | |||
| align="center" style="background:#f0f0f0;"|'''Reaction''' | |||
| align="center" style="background:#f0f0f0;"|'''1 (CMV)''' | |||
| align="center" style="background:#f0f0f0;"|'''2 (CMV)''' | |||
| align="center" style="background:#f0f0f0;"|'''3 (HPK)''' | |||
| align="center" style="background:#f0f0f0;"|'''4 (HPK)''' | |||
|- | |||
| 20 fmol of each DNA part (µL)||1+1||1+1||1+1|| | |||
|- | |||
| 10x T4 ligase buffer (Promega) (µL)||1||1||1|| | |||
|- | |||
| T4 ligase (NEB) (µL)||0.25||0.25||0.25||0.25 | |||
|- | |||
| BSMBI (µL)||0.5||0.5||0.5||0.5 | |||
|- | |||
| dH2O (µL)||6.25||6.25||6.25||6.25 | |||
|- | |||
| Total (µL)||10||10||10||10 | |||
|} |
Revision as of 16:51, 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
Backbone vector [math]\displaystyle{ X= (4600 / 209 ) * 0.013 * 20 = 5.72 }[/math] HPK [math]\displaystyle{ X= (2203 / 70 ) * 0.013 * 20 = 8.18 }[/math] CMV [math]\displaystyle{ X= (2275 / 42 ) * 0.013 * 20 = 14.08 }[/math]
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