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Revision as of 21:45, 10 September 2015

Valencia UPV iGEM 2015

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%

FOTO

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; Ω1Etr8 CMV+Bxb1_T35S; α1
1µL (GB892) PIF; α11µL 1097 (Etr8 CMV) pUPD2
1µL (GB88E) PhyB; α21µL Bxb1; pUPD2
1µL Ω1 1µL Tnos PuPD
1.2µL Buffer ligase1µL α1
1µL Bsmb15.8µL H2O
6.8µL H2O

Digestions:

(GB160) 35S:Renilla:tNOS-35S:P19:tNOSEcoRV2475, 381, 4601
(GB896) Luc:PIF6:PhyBEcoRV11608, 3942

6 June 2015

Transform to E. coli from PIF+Phy and BxbI and make petri dish cultures.

Digest of 160, 289 and the two ligations, PIF+phy and Etr8+BxbI.

Agarose gel.

GB160289PIF+PhyBBxbI
okno??

FOTO


7 June 2015

We?ve got white colonies from PIF+Phy and Bxb1!

Pick two colonies from each construction.


8 June 2015

Minipreps of the 4 liquid cultures and digestion to see the band patterns.

Digestion:

Etr8(CMV):Bxb1:Tnos; α1EcoRI6345, 238
EPIF6 + PhyB-PV16; Ω1BamHI6686, 1439, 2685, 2237

Agarose gel was made:

Bxb1 (C1)Bxb1 (C2)EPIF6 + PhyB-PV16 (C1)EPIF6 + PhyB-PV16 (C2)
okok

FOTO

Repeat digestion because we are not sure of the last digestions.

We don?t have sure the toggle, so we decide to repeat the digestion with other enzyme tomorrow, noticing that the colony 2 has better bands pattern.

Optimized ligation:

PIF-Phy-Luc-Renilla-P19
1 µL vector
0.8 µL dilution ½ GB160
1.7 µL PIF:PhyB
4.15 µL H2O
Ratio 1:2 vector insert

As BxbI was good at the digestion we put 1 µL of LB and 1 µL of Kanamicyne on the tube where it had grown and store at 37ºC to glycerinate later.

We design primers to binding domain (BD) and PIF.

  • Problem: domesticator is introduced in an old pUPD2. The new one has different bases.
  • Change manually the pUPD2 bases in the program (Benchling).

9 June 2015

Digestion of the ligation of yesterday containing: EPIF6-PhyB-VP16 (C1 y C2)

EPIF6-PhyB-VP16PvuII (green buffer)3663, 9472pb

Agarose gel 1%:

EPIF6-PhyB-VP16 (C1)EPIF6-PhyB-VP16 (C2)
nono

FOTO

We see three bands: 7000, 4000, 1900pb

Transform optimized ligation PIF-Phy-Luc-Renilla-P19 and make petri dish cultures.