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Revision as of 11:25, 28 August 2015

Valencia UPV iGEM 2015

The project
Notebook


Notebook

Contents:


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%

MwPles4E4B3E3B 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; ?1Etr8 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 ligase1µL α1
1µL Bsmb15.8µL 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 ladder160289ligationligationLadder
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; Ω1EcoRI6345, 238
EPIF6 + PhyB-PV16; Ω1BamHI6686, 1439, 2685, 2237