Team:MIT/miRNA Group Notebook TRE Dox
Dox Curve 4.0
Made with Benchling
Project: miRNA and Repressors subgroup
Authors: Elizabeth Strand
Date: 2016-08-09
Tuesday, 8/9
Changes to this DOX curve attempt: Recieved different DOX solution tubes from Brian and also increasing rtTA and TRE ration from 1:3 to 1:1
Dox Tubes from Brian: 100uM = 100,000nM. Made 5,000nM dilution as well (10 uL 100,000 stock in 195uL H20)
using equation C1*V1=C2*V2
General procedure: Creating 500 uL Dox media aliquots of to induce transfected HEK293 cells with DOX concentrations 20nM, 50nM, 100 nM, 200nM, 500nM, 1000nM, and 2000 nM.
During transfection, aspirate exisiting media, replace with 450 uL of Dox media, and then add 50 uL vifect transfection complex.
Harvesting for Flow Cytometry will occur Friday.
A | B | C | D | E | F | G | |
1 | Stock Concentration (nM) | Concentration in Dox Media Solution (nM) | Dox Media Final Volume (uL) | How Much Dox Stock Concentration to Add to Media (uL) | Media to Add | Amount of Final Dox Solution to Add to Well (uL) | Final Dox Concentration in Well (nM) |
2 | 5000 | 22 | 500 | 2.2 | 497.8 | 450 | 20 |
3 | 5000 | 55 | 500 | 5.5 | 494.5 | 450 | 50 |
4 | 5000 | 110 | 500 | 11 | 489 | 450 | 100 |
5 | 5000 | 220 | 500 | 22 | 478 | 450 | 200 |
6 | 100000 | 550 | 500 | 2.75 | 497.25 | 450 | 500 |
7 | 100000 | 1100 | 500 | 5.5 | 494.5 | 450 | 1000 |
8 | 100000 | 2200 | 500 | 11 | 489 | 450 | 2000 |
Table1
Plasmids Used (all concentrations about 100ng/ul):
TRE:BFP
hef1a:mKate
hef1a:BFP
hef1a:rtTA
pDONR_GG (blank DNA)
Experimental Wells:
3 uL TRE:BFP
1 uL hef1a:mKate
3 uL hef1a:rtTA
3 uL pDONR
2 uL viafect
38 uL Opti-MEM
450 uL DOX Media
Single Color Wells:
1 uL hef1a: color (BFP or mKate)
9 uL pDONR_GG
2 uL viafect
38 uL Opti-MEM
450 uL Media
Two Color Well:
1 uL hef1a:BFP
1 uL hef1:mKate
8 uL pDONR_GG
2 uL viafect
38 ul Opti-MEM
450 uL Media
Dox2.PNG
A | B | C | |
1 | Well | Sample | Dox (nM) |
2 | 1 | Experimental | 0 |
3 | 2 | Experimental | 20 |
4 | 3 | Experimental | 50 |
5 | 4 | Experimental | 100 |
6 | 5 | Experimental | 200 |
7 | 6 | Experimental | 500 |
8 | 7 | Experimental | 1000 |
9 | 8 | Experimental | 2000 |
10 | 9 | 2 color | n/a |
11 | 10 | mKate | n/a |
12 | 11 | BFP | n/a |
13 | 12 | untransfected | n/a |
Table2
Experimental Set Up (Old)
Made with Benchling
Project: miRNA and Repressors subgroup
Authors: Wangui Mbuguiro
Date: 2016-07-14
Thursday, 7/14
Dox serial dilution done in HEK 293 media
20160712_180815.jpg
450 uL of of each dilution was added to each well and then 50 uL od
Calculation of final concentrations
A | B | C | D | E | F | |
1 | Conc. in 450 uL (ng/mL) | 1000 | 200 | 50 | 10 | 2 |
2 | Final Conc (in 500 uL well) (ng/mL) | 900 | 180 | 45 | 9 | 1.8 |
Table1
Well set up
20160712_191100.jpg
Transfection to characterize TRE
Made with Benchling
Project: miRNA and Repressors subgroup
Authors: Wangui Mbuguiro
Date: 2016-07-12
Tuesday, 7/12
A | B | C | D | E | |
1 | Construct | Conc. (ng/uL) | Diluted Stock: | Volume to get 500 ng total | |
2 | pDONR (in tissue culture) | 200 | - | ||
3 | TRE:BFP | 765.6 | 100 | 1 | |
4 | hef1a:rtTA | 133.4 | - | 2.5 | |
5 | hef1a:BFP | 610.2 | 100 | ||
6 | hef1a:EYFP | 521.3 | 100 | 1 |
Table1
Dox: 1 ug/uL
