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
|
Sample
|
Primer |
|
1 and 2
|
iGEM2018_60/61 |
|
3 and 4
|
iGEM2018_62/63 |
|
5 and 6
|
iGEM2018_64/65 |
|
7 and 8
|
iGEM2018_66/67 |
|
9
|
negative control |
DNA isolated from the plates by pipetting 10 µL dH2O.
PCR samples were purified using the QIAquick PCR purification Kit (250).
Nanodrop measurement
sample
|
concentration [ng/µL]
|
| BFP |
26.0
|
| GFP |
13.1
|
| mCherry |
10.7
|
| mOrange |
24.0
|
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 |
|
Nanodrop measurement
sample
|
concentration [ng/µL]
|
| BFP |
5.8
|
| GFP |
14.0
|
| mCherry |
10.6
|
| mOrange |
15.8
|
| pAC7 |
4.0
|
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
