Difference between revisions of "Team:Goettingen/Notebook/fluorophores July"
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Latest revision as of 11:13, 15 October 2018
Contents
Fluorophore experiments
30.07.18
PCR of Biobricks
| Primer for BFP | Position on plate |
|---|---|
| iGEM2018_60 | 17 I Plate1 |
| iGEM2018_61 | |
| Primer for GFP | Position on plate |
| iGEM2018_62 | 4B Plate 1 |
| iGEM2018_63 | |
| Primer for mOrange | Position on plate |
| iGEM2018_64 | 1D Plate 5 |
| iGEM2018_65 | |
| Primer for mCherry | Position on plate |
| iGEM2018_66 | 11P Plate 6 |
| iGEM2018_67 |
PCR Set-up
|
|
Primer |
|---|---|
|
|
iGEM2018_60/61 |
|
|
iGEM2018_62/63 |
|
|
iGEM2018_64/65 |
|
|
iGEM2018_66/67 |
|
|
negative control |
DNA isolated from the plates by pipetting 10 µL dH2O.
PCR samples were purified using the QIAquick PCR purification Kit (250).
sample |
|
|---|---|
| BFP |
|
| GFP |
|
| mCherry |
|
| mOrange |
|
For downstream analysis, and to isolate the fluorescence proteins, a restriction digest was carried out with BFP, GFP, mOrange, and mCHerry.
Reaction mixtures to digest the fragments
| Compound | Volume in [µL] |
|---|---|
| BFP | 11.5 |
| EcoRII | 2 |
| BamHI | 2 |
| FD buffer | 3 |
| H2O | 21.5 |
| Incubate for 30 min at 37°C. The reation was stopped using the PCR purification kit |
| Compound | Volume in [µL] |
|---|---|
| GFP | 23 |
| EcoRII | 2 |
| BamHI | 2 |
| FD buffer | 3 |
| H2O | 10 |
| Incubate for 30 min at 37°C. The reation was stopped using the PCR purification kit |
| Compound | Volume in [µL] |
|---|---|
| mOrange | 12.5 |
| EcoRII | 2 |
| BamHI | 2 |
| FD buffer | 3 |
| H2O | 21.5 |
| Incubate for 30 min at 37°C. The reation was stopped using the PCR purification kit |
| Compound | Volume in [µL] |
|---|---|
| mCherry | 30 |
| EcoRII | 2 |
| BamHI | 2 |
| FD buffer | 3 |
| H2O | 3 |
| Incubate for 30 min at 37°C. The reation was stopped using the PCR purification kit |
sample |
|
|---|---|
| BFP |
|
| GFP |
|
| mCherry |
|
| mOrange |
|
| pAC7 |
|
The strains SP1, BP233, BP235 and SPden were propagated to 4 mL LB medium and grown at 28°C over night to make them competent