User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/11/06: Difference between revisions
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* Add 1 μL FastDigest DpnI and 5 μL of 10x FastDigest buffer to each PCR reaction. PCR product. Incubate at 37°C for 15 min. Note: DpnI cuts the methylated template DNA, not the unmethylated | |||
{| {{table}} | |||
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| bgcolor="grey" | Reagent | |||
| bgcolor="grey" | Vol. | |||
| rowspan=7 | '''DpnI Digest''' | |||
* 37°C, 15 min. | |||
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| PCR reaction || 95.0 μL | |||
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| 10x FD buffer (Fermentas) || 5.0 | |||
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| DpnI (Fermentas) || 1.0 | |||
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| dH<sub>2</sub>O || --- | |||
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| || 102.0 μL | |||
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Revision as of 18:04, 6 November 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= (5500 / 170 ) * 0.013 * 20 = 8.4 + 11.58 = 20 }[/math]μL HPK [math]\displaystyle{ X= (2203 / 115 ) * 0.013 * 20 = 1.16 + 19 = 20 }[/math]μL CMV [math]\displaystyle{ X= (2275 / 55 ) * 0.013 * 20 = 2.78 + 17.2 = 20 }[/math]μL
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