User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/06/06: Difference between revisions
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* ✓ Transfections: | * ✓ Transfections: H3K27me3 reporters into cell lines BD002 (KAH201+KAH182+V0200) | ||
* ✓ Polycomb-ATF negative control lines: colony plates | * ✓ Polycomb-ATF negative control lines: colony plates | ||
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---- | ---- | ||
'''Transfections'''<br> | '''Transfections'''<br> | ||
> U2OS Flp-in T-REx lines (puro/zeo/blast) + | > U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)<br> | ||
--> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in<br> | --> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in<br> | ||
> Use Lipofectamine, 6-well format<br> | > Use Lipofectamine, 6-well format<br> | ||
> Plates: | > Plates: | ||
# | # BD002 (3:1) Flp-in T-REx | ||
# | #BD002 (4:1) Flp-in T-REx | ||
BD002 (3:1) = 339 ng/μl | |||
BD002 (4:1) = 317 ng/μl | |||
PlpE = 192 ng/μl | |||
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| <u>Plate-Well</u> || <u>Plasmid</u> || <u>DNA</u> || <u>Volume</u> || <u>Lipo</u> || <u>Opti-MEM (total)</u> | | <u>Plate-Well</u> || <u>Plasmid</u> || <u>DNA</u> || <u>Volume</u> || <u>Lipo</u> || <u>Opti-MEM (total)</u> | ||
|- | |- | ||
| 1-1 || | | 1-1 || BD002 (3:1) + FlpE || 1.5 + 0.5 μg || 4.42 + 2.60 μL || 4 μL || 500 μL | ||
|- | |- | ||
| 2 | | 1-2 || BD002 (3:1) + FlpE || " || 4.42 + 2.60 μL || " || " | ||
|- | |- | ||
| | | 1-3 || BD002 (4:1) + FlpE || " || 4.73 + 2.60 μL || " || " | ||
|- | |- | ||
| | | 1-4 || BD002 (4:1)+ FlpE || " || 4.73 + 2.60 μL || " || " | ||
|- | |- | ||
| | |1-5 || BD002 (3:1) + --- || 4.42 + --- || || | ||
|- | |- | ||
|1-6 || BD002 (4:1) + --- || 4.73 + --- || || | |||
| --- | | --- | ||
> Add 16 μL (4x) Lipo to 1000 μL (4x) Opti-MEM --> R.T/ 5 min. <br> | > Add 16 μL (4x) Lipo to 1000 μL (4x) Opti-MEM --> R.T/ 5 min. <br> |
Revision as of 11:43, 6 June 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> | |||||||||||||||||||||||||||||||||||||||||
06/06/2013
Transfections
BD002 (3:1) = 339 ng/μl BD002 (4:1) = 317 ng/μl PlpE = 192 ng/μl
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