June 24th, 2010
1. Inactivate restriction enzymes from June 23rd restriction, 80ºC for 20 min.
2. Run gel to verify extracted plasmids and restricted plasmids GFP reporter (E0240) and backbone plasmid (pSB3K3).
Lanes: 1) Ladder; 2) GFP E0240 1; 3) GFP E0240 2; 4) Backbone pSB3K3 1; 5) Backbone pSB3K3 2; 6) XbaI-PstI GFP E0240; 7) EcoRI-PstI Backbone pSB3K3.
3. Make PCR to amplify p30 and join with Minimum Blue Promoter.
- I’ll do it with my reactives and Paz’s.
- Enzyme used: rTth
- Tubes are marked 1-6:
- p30-MinBP Jorge’s reactives
- Ctrl ~3 kb’s Jorge’s reactives
- Ctrl - Jorge’s reactives
- p30-MinBP Paz’s reactives
- Ctrl ~3 kb’s Paz’s reactives
- Ctrl - Paz’s reactives
- Reactives needed for one reaction:
- PCR with RTTH polymerase
- Reaction 1 (ul x sample)
- HPLC -> 6
- Buffer 3.3x -> 6
- Mg(Ac)2 -> 3
- dNTP’s -> 5
- Primer FWD -> 3
- Primer REV -> 3
- DNA -> 4
- Total volume -> 30
- Reaction 2 (ul x sample)
- HPLC -> 10.5
- Buffer 3.3x -> 9
- RTTH -> 0.5
- Total -> 20
4. Incubate transformed cells with backbone plasmid to extract plasmid tomorrow.
- Advances and perspectives
- We finnaly could insert the minimum blue promoter into plasmid 30 pSB4A5 by PCR.
- This PCR product will be purified and GFP reporter will be inserted into this PCR product.
- As GFP reporters we’ll use GFP E0240, recommended in the BioBrick Promoter Measurement Kit, and also GFP BBa_K145015, this reporter is well characterized by the KU Leuven 2008 team and has a half-life of 74 minutes.
- To test Blue Promoter functionality I’ll use the iGEM BioBrick Promoter Measurement Kit. For this test I’ve done transfromations, plasmid extraction and restrictions to the GFP reporter (E0240), a backbone plasmid (pSB3K3) and BBa_I20260, a GFP regulated under the constitutive promoter J23101, as reference promoter.
- Ligation with Blue Promoter + GFP reporters (the two GFPs described above) + Backbone plasmid will be performed by three different ways:
- First ligate Blue Promoter + GFP reporters, and then ligate this construct to the backbone plasmid.
- Blue Promoter + GFP reporters + Backbone plasmid by a 3-way ligation
- Insert the Minimum Blue Promoter to backbone plasmid by PCR and then ligate GFP reporters to this PCR product.
- I’ve done the transformations with a YFP reporter BBa_K117008 that will be used for the fluorescence assay.