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
Dox Curve 2.0
Made with Benchling
Project: miRNA and Repressors subgroup
Authors: Elizabeth Strand
Date: 2016-07-29
Friday, 7/29
*seeded 24 well plate 24 hours before transfection (150,000 cells/well)
DoxCurve2Real.PNG
*Conclusion: Dox saturation occurs at about 50 to 100nM. Assuming DOX stock given to us is not 1g/L and that the concentration it magnitudes higher. Will attempt to nanodrop. Brian is making new stock.
DOX Stock from Brian: 1 gram DOX/ Liter = 2,252,253 nanoMolar
Molecular weight of DOX: 444grams
DOX and Media Solution created for DOX concetrations in TRE:BFP Dox Curve experiment (HEK293)
*made under tissue culture hood
A | B | C | D | |
1 | [DOX] | Amount Brian Stock | Amount Media | Total Volume |
2 | 20,000 nM | 13.3 uL | 1486.7 uL | 1.5 mL |
3 | 5,000 nM | 3.4 uL | 1496.6 uL | 1.5 mL |
4 | Amount 20,000nM Stock | Amount of Media | ||
5 | 2,000 nM | 50 uL | 400 uL | 450 uL |
6 | 1,000 nM | 25 uL | 425 uL | 450 uL |
7 | 500 nM | 12.5 uL | 437.5 uL | 450 uL |
8 | 200 nM | 5 uL | 445 uL | 450 uL |
9 | 100 nM | 2.5 uL | 447.5 uL | 450 uL |
10 | Amount of 5,000 nM Stock | Amount of Media | ||
11 | 50 nM | 5 uL | 445 uL | 450 uL |
12 | 20 nM | 2.5 uL | 447.5 uL | 450 uL |
13 |
Table1
Dox2.PNG
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
1 uL hef1a:rtTA
1 uL viafect
44 uL Opti-MEM
450 uL DOX Media
Single Color Wells:
1 uL hef1a: color (BFP or mKate)
3 uL pDONR_GG
1 uL viafect
44 uL Opti-MEM
450 uL Media
Two Color Well:
1 uL hef1a:BFP
1 uL hef1:mKate
2 uL pDONR_GG
1 uL viafect
44 ul Opti-MEM
450 uL Media
Dox Curve 3.0
Made with Benchling
Project: miRNA and Repressors subgroup
Authors: Elizabeth Strand
Date: 2016-08-02
Tuesday, 8/2
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
1 uL hef1a:rtTA
1 uL viafect
44 uL Opti-MEM
450 uL DOX Media
Single Color Wells:
1 uL hef1a: color (BFP or mKate)
3 uL pDONR_GG
1 uL viafect
44 uL Opti-MEM
450 uL Media
Two Color Well:
1 uL hef1a:BFP
1 uL hef1:mKate
2 uL pDONR_GG
1 uL viafect
44 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
