User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/08/08: Difference between revisions
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> U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)<br> | > U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)<br> | ||
--> Note: co-transfect with | --> Note: BD002 co-transfect with Gal4-VP64 (10:1); hygro resistance replaces zeo resistance after transfection<br> | ||
> Use Lipofectamine, 6-well format<br> | > Use Lipofectamine, 6-well format<br> | ||
> | > Plasmids: | ||
#BD002 | # BD002 = 346.61 ng/μl | ||
# [http://openwetware.org/wiki/User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli/2013/08/05 Gal4-VP64 transactivation plasmids] | |||
## KAH59 (MV5) = 277.719 | |||
##KAH54 (PCVN) = 217.267 | |||
##KAH59 (PCVN) = 203.300 | |||
##KAH60 (PCVN) = 169.931 | |||
##KAH66 (MV5) = 174.819 | |||
{| {{table}} cellspacing="7" width=700px | {| {{table}} cellspacing="7" width=700px | ||
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| Plate-Well || Plasmid || DNA || Volume|| dH2O|| Lipo || PLUS reagent || <u>Opti-MEM (total)</u> | | Plate-Well || Plasmid || DNA || Volume|| dH2O|| Lipo || PLUS reagent || <u>Opti-MEM (total)</u> | ||
|- | |- | ||
| 1-1 || BD002 ( | | 1-1 || BD002 + KAH59(PcVN) || 1.81 + 0.19 μg || 5.23 + 0.89 μL || 13.88μL|| 2.5μL|| 7.5 μL || 570 μL | ||
|- | |- | ||
| 1-2 || BD002 ( | | 1-2 || BD002 + KAH60(PcVN) || " || 5.23 + 1.07 μL || 13.7μL|| " || " || " | ||
|- | |- | ||
| 1-3 || BD002 ( | | 1-3 || BD002 + KAH54(PcVN) || " || 5.23 + 0.83 μL || 13.94μL || " || " || " | ||
|- | |- | ||
| 1-4 (-)Ctrl || BD002 ( | | 1-4 (-)Ctrl || BD002 + KAH59(MV5) || " || 5.23 + 0.65 μL || 14.12μL|| " || " || " | ||
|- | |- | ||
|1-5 (-) Ctrl || | |1-5 (-) Ctrl || BD002 + KAH66(MV5) || " || 5.23+1.04 || 13.73μL|| " || "||" | ||
|- | |- | ||
|} | |} | ||
Revision as of 12:46, 9 August 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> | ||||||||||||||||||||||||||||||||||||||||||||||||
08/08/2013 Gal4-VP64, Cyan Transactivation1 day before transfection:
Transfections > U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)
Transgene expression should be detectable after 18 hours.
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