Team:Valencia UPV/Notebook
Constructions
CONSTRUCTIONS
5 June 2015
We had 2 cultures from the last day, corresponding to other 2 colonies of ligation.
Agrobacterium culture of promoter less: Luciferase + Renilla
Minipreps
Digestion with BamHI and EcoRV
Agarose gel 1%
| Mw | Ples | 4E | 4B | 3E | 3B 1kb |
How to ask and make primers?
- Select the sequence to amplify and save in FASTA format.
- gbCloning, go to Tools-Domesticator-1º Category
- Add FASTA and select parts.
- On the protocol we have the primers
- The oligos they give us:
- 4 first nucleotides: so the enzyme can recognize without problems
- 6 following bingind sites.
- 1 extra nucleotide.
- 4 overhangs.
Meeting with Daniel Ramón (Biopolis).
Ligation with part 2 and 24 of task sheet.
| Pif6 + PhyB; ?1 | Etr8 CMV_Bxb1_T35S |
| 1µL 892 (Pif α1) | 1µL 1097 (Etr8 CMV) Pupd2 |
| 1µL 88E (Phy α2) | 1µL Bxb1 (PuPD) |
| 1µL ?1 | 1µL Tnos PuPD |
| 1.2µL Buffer ligase | 1µL α1 |
| 1µL Bsmb1 | 5.8µL H2O |
| 6.8µL H2O |
If we make a digestion of 160 (35S:Renilla:tNOS-35S:P19:tNOS) with EcoRV, we obtain: 2475, 381, 4601 pb.
If we make a digestion of 896 (Luc:Pif6:PhyB)with EcoRV, we obtain: 11608, 3942 pb.
June 2015
Transform to E.coli from Pif+Phy and Bxb1
- 1.5µL of ligation
- Cuvette on ice
- Competent cells + 1.5µL of ligation
- Pulse (electroporator) at 1500V
- Add 300µL shock medium and put Eppendorf 1h at 37ºC
Culture on petri dishes the ligations.
Digest of 160, 289 and the two ligations, pif+phy and Etr8+BxbI.
Agarose gel.
| 6µL ladder | 160 | 289 | ligation | ligation | Ladder |
| 1Kb |
Storage of gel on: Basura en Arabidopsis – Igem – 2015 – 150606_Digestion_ToggleRojo
7 June 2015
We’ve got white colonies! (from Pif+Phy and Bxb1)
Pick two colonies from each construction.
4 tubes
- 3.5µL LB each tube
2) 2 tubes + 3.5µL Kanamycin (K)
8 June 2015
Minipreps of the 4 liquid cultures and digestion to see the band patterns.
Digestion:
| Etr8(CMV):Bxb1:Tnos; Ω1 | EcoRI | 6345, 238 |
| EPIF6 + PhyB-PV16; Ω1 | BamHI | 6686, 1439, 2685, 2237 |