# User:Karmella Haynes/Notebook/PcTF Genomics/2013/07/04

(Difference between revisions)
 Revision as of 00:04, 5 July 2013 (view source) (→07/04/13)← Previous diff Revision as of 00:11, 5 July 2013 (view source) (→07/04/13)Next diff → Line 58: Line 58: | sample 6 || 17,885  || x5 = || 89,425 | sample 6 || 17,885  || x5 = || 89,425 |} |} + + + + ---- + '''Gal4-EED/luc Dox induction #2'''
+ * Lower range worked very well for trail #1! Saw ranges in expression for 1.0 - 10-4 μg/mL doc after 4 days + * Repeat induction and test after 24 hours + + # 1.0 μg/mL dox + # 10-1 + # 10-2 + # 10-3 + # 10-4 + # 0 + + Addition of serial dilutions of dox to cells + * Start with 3.5 mL growth medium in cell culture wells. Remove some growth medium from cells where necessary. + * Set up the following serial dilutions (using appropriate growth medium) in sterile 1 mL tubes... + + {| {{table}} + |- + |   || tube 1 || 2 || 3 || 4 || 5 || 6 + |- + | 1 mg/mL dox (μL) || 8.0 || --- || --- || --- || --- || --- + |- + | mixture from prior tube (μL) || --- || 100 || 100 || 100 || 100 || --- + |- + | +growth med. (μL) || 1000 || 900 ||  900 ||  900 ||  900 || --- + |- + | Final [dox] (μg/mL) in 4 mL med. || 1.0 || 10-1 || 10-2 || 10-3 || 10-4 || 0 + |} +

## Revision as of 00:11, 5 July 2013

Pc-TF Genomics Main project page
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## 07/04/13

• Gal4-EED/luc time/dose - luc assay & cell counts
• Happy July 4th! :)
• Gal4-EED/luc Dox induction #2

Luciferase activity assay - time point: 4 days

Cell prep

• Harvested cells (induced on 6/30/13)
• Seeded new 2 new plates with non-induced cells from 6/30/13
• Pelleted all of the dox+ cells, resuspended in 2 mL FACS buffer

Assay reagents

• Used the Biotium Steady-Luc Firefly HTS Assay Kit (same as 6/18/13)

Luc assay

• Filtered 700 μL cells through strainer caps
• Used opaque white Costar plate (from Rege lab)
• Samples loaded in triplicate (by columns)
• Included luc buffer + FACS-buffer "blank" sample (well D1)
• Other steps same as 6/18/13

Cell counts

• Wang lab's Accuri flow cytometer.
• Set machine to read 20 uL of cells
• Be sure to "clean" with water-run in between samples

 Sample ID Gated count/ 20 μL Cells/ 100 μL sample 1 13,844 x5 = 69,220 sample 2 12,286 x5 = 61,430 sample 3 15,784 x5 = 78,920 sample 4 16,323 x5 = 81,615 sample 5 16,545 x5 = 82,725 sample 6 17,885 x5 = 89,425

Gal4-EED/luc Dox induction #2

• Lower range worked very well for trail #1! Saw ranges in expression for 1.0 - 10-4 μg/mL doc after 4 days
• Repeat induction and test after 24 hours
1. 1.0 μg/mL dox
2. 10-1
3. 10-2
4. 10-3
5. 10-4
6. 0

Addition of serial dilutions of dox to cells

• Start with 3.5 mL growth medium in cell culture wells. Remove some growth medium from cells where necessary.
• Set up the following serial dilutions (using appropriate growth medium) in sterile 1 mL tubes...
 tube 1 2 3 4 5 6 1 mg/mL dox (μL) 8.0 --- --- --- --- --- mixture from prior tube (μL) --- 100 100 100 100 --- +growth med. (μL) 1000 900 900 900 900 --- Final [dox] (μg/mL) in 4 mL med. 1.0 10-1 10-2 10-3 10-4 0