User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/17: Difference between revisions
From OpenWetWare
Line 85: | Line 85: | ||
| align="center" style="background:#f0f0f0;"|'''3 (HPK)''' | | align="center" style="background:#f0f0f0;"|'''3 (HPK)''' | ||
| align="center" style="background:#f0f0f0;"|'''4 (HPK)''' | | align="center" style="background:#f0f0f0;"|'''4 (HPK)''' | ||
| align="center" style="background:#f0f0f0;"|'''Thermal Cycling Reaction Conditions''' | |||
|- | |- | ||
| 20 fmol of each DNA part (µL)||1+1||1+1||1+1|| | | 20 fmol of each DNA part (µL)||1+1||1+1||1+1|| 1+1 || rowspan='6' | | ||
* [45°C, 2 min.; 16°C 5 min.] x25 | |||
* 60°C, 10 min. | |||
* 80°C, 20 min. | |||
*4°C, ∞ | |||
|- | |- | ||
| 10x T4 ligase buffer (Promega) (µL)||1||1||1|| | | 10x T4 ligase buffer (Promega) (µL)||1||1||1|| 1 | ||
|- | |- | ||
| T4 ligase (NEB) (µL)||0.25||0.25||0.25||0.25 | | T4 ligase (NEB) (µL)||0.25||0.25||0.25||0.25 | ||
Line 98: | Line 103: | ||
| Total (µL)||10||10||10||10 | | Total (µL)||10||10||10||10 | ||
|} | |} | ||
* Bacterial transformation , Long transformation protocol | |||
** Add total volume (10.0 μL) to 50 μL BL21 and DH5α, Incubate on ice for 1 min., heat shock at 42°C for exactly 45 sec., immediately place on ice for 1 min. Add 800 μL sterile SOC medium. Grow with shaking at 37°C for 45 min. Pellet the cells for 3 min. at room temp. Resuspend the cells in 100 μL LB + AMP antibiotic. Plate cells on pre-warmed LB agar + AMP antibiotic. Grow overnight at 37°C. |
Revision as of 16:58, 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]
|