User:Karmella Haynes/Notebook/Polycomb project/Entry Base: Difference between revisions

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Line 13: Line 13:
> HEK293 cells + Gal4/mCh/VP64 constructs<br>
> HEK293 cells + Gal4/mCh/VP64 constructs<br>
> Use Fugene, 24-well format<br>
> Use Fugene, 24-well format<br>
> Dilute each plasmid to 100 ng/ul in st dH2O<br>
> Dilute each plasmid to 100 ng/ul in st dH<sub>2</sub>O<br>


{| class="wikitable" border="0" cellspacing="5"
{| class="wikitable" border="0" cellspacing="5"
Line 27: Line 27:
| KAH59/VNpc
| KAH59/VNpc
| 25-800 ng
| 25-800 ng
| 0.3, 0.5, 1.0, 2.0, 4.0, 8.0
| 0.3, 0.5, 1.0, 2.0, 4.0, 8.0 ul
| 1.8 ul
| 1.8 ul
| 18.2 ul
| 18.2 ul

Revision as of 07:55, 19 November 2009

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page

11/19/09

  • Transfections: test Gal4/mCh/VP64 ATF's (trial 2)


Transfections
> HEK293 cells + Gal4/mCh/VP64 constructs
> Use Fugene, 24-well format
> Dilute each plasmid to 100 ng/ul in st dH2O

Wells Construct DNA Volumes Fugene Opti-MEM
1-6 KAH59/VNpc 25-800 ng 0.3, 0.5, 1.0, 2.0, 4.0, 8.0 ul 1.8 ul 18.2 ul
7-12 KAH60/VNpc " " " "
13-18 KAH54/VNpc " " " "
19-24 KAH66/MV5 " " " "
25-30 KAH65/MV5 " " " "
31-36 KAH59/MV5 " " " "
37-42 KAH36 " " " "
43-48 none " " " "

> Fugene + Opti-MEM --> R.T/ 5 min.
> Add DNA --> R.T./ 20 min.
> Add complexes to each well (1 ml med. each); 37
> Assay RFP/ Luc after 18-24 hours



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