Take glycerinated cultures of C58 Agrobacterium with dsRED from GoldenBraid Collection. Prepare broth culture (5 mL LB + 5 μL kanamycin + 5 μL Rifampin) 1:1000 and incubate overnight at 28°C.
19/05/2016
Refresh previously made culture by inoculating 10 μL in a new culture medium.
20/05/2016
Agroinfiltration in Nicotiana benthamiana of C58 with dsRED.
06/06/2016
Take from the glycerinates of Goldenbraid Collection:
Plasmid
GB Code
pD6B3 α1
GB0015
pD6B3 α2
GB0017
pD6B3 Ω1
GB0019
pD6B3 Ω2
GB0021
pUPD2
GB0307
Prepare liquid culture (3 mL LB + 3 μL antibiotic) 1:1000. Incubate at 37°C overnight.
Experiment with snails:
Let both N. benthamiana leafs with snails overnight at room temperature in separated boxes. We will observe if snails eat the leafs and if appears fluorescence.
15/06/2016
Experiment is over due to snails haven’t eaten leafs enough so we have not been able to see fluorescence.
30/06/2016
Orange DNA Genome Extraction protocol
Take from the glycerinates of GoldenBraid Collection:
gBlocks of - promoter 35s:5’ region - have arrived.
We perform a PCR of orange and rice Genome Extraction following the protocol
Sample
Initial concentration(ng/μL)
Final concentration(ng/μL)
Initial volume(μL)
Final volume (μL)
Clemenules 1
3153.8
150
4.756
100
Clemenules 2
4527.9
150
3.31
100
Gleva 1
294.9
150
50.8647
100
Gleva 2
193.7
150
77.44
100
Reagent
Volume(μL)
Program
Clemenules DNA 1
1
Temperature
Time
Buffer HF
10
98°C
5 minutes
dNTPs
2
98°C
35x
30 seconds
IG16JUL01 (TFL_For)
2.5
64°C
30 seconds
IG16JUL02 (TFL_Rev)
2.5
72°C
30 seconds
Taq phusion
0.5
72°C
10 minutes
H2O milli-Q
31.5
16°C
∞
Reagent
Volume(μL)
Program
Gleva DNA
1
Temperature
Time
Buffer HF
10
98°C
5 minutes
dNTPs
2
98°C
35x
30 seconds
IG16JUL03 (Ga20_for)
2.5
72°C
30 seconds
IG16JUL02 (Ga20_rev)
2.5
72°C
30 seconds
Taq phusion
0.5
72°C
10 minutes
H2O milli-Q
31.5
16°C
∞
Ligate reaction of promoter 35s:5’ region in pUPD2. Following ligation protocol , BsmbI enzyme is used in this reaction.
08/07/2016
Run electrophoresis gel of Clemenules and Gleva PCR products. 45 mL agarose gel at 1 % 0.45 g of agarose with 45 mL of TAE 1X. Voltage used is 120 V.
Transform E. coli with the next devise: promoter 35s:5’ region (electroporation 2.5KV). Plating it and incubate overnight at 37°C.
Take glycerinated culture for Georgia collaboration. The devise is promoter 35s:GFP:Tnos (α1 and kanamycin)
09/07/2016
Miniprep with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
promoter 35s:GFP:Tnos
No colonies have grown in the devise promoter 35s:5’ region petri dishes. Repeat transformation procedure, plating again and incubate overnight at 37°C.
11/07/2016
Pick a single E. coli DH5α (promoter 35s:5’ region in pUPD2) colony from the plate that has been incubated overnight. Inoculate a starter culture of 4 ml of LB medium with 4 μL of chloramphenicol in a 50 ml tube with the colony and incubate it overnight at 37°C with shaking.
Check Georgia miniprep concentration with NanoDrop (promoter 35s:GFP : Tnos)
Sample
DNA Concentration(ng / μL)
DNA Concentration(ng)
1
105.2
5035.2
2
104.6
5035.2
Targets ligations in pUPD2 (Orange Clemenules and Rice Gleva). Following ligation protocol , BsmbI enzyme is used in this reaction.
12/07/2016
Pick a single E. coli DH5α (target Gleva in pUPD2 and target Clemenules in pUPD2) colony from the plate that has been incubated overnight. Inoculate a starter culture of 4 ml of LB medium with 4 μL of chloramphenicol in a 50 ml tube with the colony and incubate it 16 hours at 37°C.
Miniprep with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
Promoter 35s:5’region in pUPD2
Digestion of minipreps with NotI. Incubate 1 hour at 37°C
Run electrophoresis gel of the following devise: promoter 35s:5’ region in pUPD2. We remain the samples 1 and 3.
Miniprep with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
Rice Gleva target in pUPD2
Orange Clemenules target in pUPD2
Digestion of minipreps with NotI. Incubate 1 hour at 37°C.
Run electrophoresis gel of the same devise:
Rice Gleva target in pUPD2
Orange Clemenules target in pUPD2
Ligation using Golden Braid assembly of the next devise:
Promoter 35s:5’ region:Target:Luc:Tnos in α1 plasmid.
13/07/2016
E. coli Transformation with the devise:
Promoter 35s:5’ region:Target:Luc:Tnos in α1 plasmid.
E. coli plating in plates with Kanamycin (antibiotic). Incubate overnight at 37°C
14/07/2016
Pick a single E. coli DH5α (promoter 35s:5’ region) colony from the plate that has been incubated overnight. Inoculate a starter culture of 4 ml of LB medium with 4 μL of chloramphenicol in a 50 ml tube with the colony and incubate it 16 hours at 37°C.
Digestion of the minipreps with EcoRI which have been done before. Incubate 1 hour at 37°C. There is a total of twelve minipreps. It has been followed the Miniprep digestion protocol .
Run electrophoresis gel of the digestion products.
Lanes
Samples
Verification
1
1 Kb molecular weight marker
2
gRNA Ga20ox 1
3
gRNA Ga20ox 2
4
Ga20ox consensus 1
5
Ga20ox consensus 2
6
Ga20ox knock-out 1
7
Ga20ox knock-out 2
8
TFL consensus 1
9
TFL consensus 2
10
TFL knock-out 1
11
TFL knock-out 2
12
TFL gRNA 1
13
TFL gRNA 2
14
1 Kb molecular weight marker
Pick a single E. coli DH5α colony from the plates that have been incubated overnight. The devises are:
Promoter 35s:TFL Knock-out:Luc:Tnos (4 samples)
Promoter U6-26:Ga20ox sgRNA:psgRNA (2 samples)
Inoculate a starter culture of 4 ml of LB medium with 4 μL of kanamycin in a 50 ml tube with the colony and incubate it 16 hours at 37°C.
Digestion of the minipreps with EcoRI which have been done before. Incubate 1 hour at 37°C. There is a total of six minipreps. It has been followed the Miniprep digestion protocol .
Run an electrophoresis gel of the digestion products.
Lanes
Samples
Verification
1
1 Kb molecular weight marker
2
gRNA Ga20ox 1
3
gRNA Ga20ox 2
4
gRNA Ga20ox 3
5
gRNA Ga20ox 4
6
TFL knock-out 1
7
TFL knock-out 2
8
1 Kb molecular weight marker
However, despite the fact that electrophoresis gel have correctly run, we have decided to repeat the ligation reactions. We have not get the expected results for a gRNA.
Ligation using Golden Braid assembly of the next devises:
Promoter 35s:5’ region:TFL Clemenules target control positive:Luc:Tnos
Promoter 35s:5’ region:Ga20ox Gleva target control positive:Luc:Tnos
Digestion of the minipreps with EcoRI which have been done before. Incubate 1 hour at 37°C. There is a total of six minipreps. It has been followed the Miniprep digestion protocol .
Run an electrophoresis gel of the digestion products. We will keep the minipreps with “1”.
Lanes
Samples
Verification
1
1 Kb molecular weight marker
2
gRNA TFL 1
3
gRNA TFL 2
4
TFL consensus 1
5
TFL consensus 2
6
TFL knock-out 1
7
TFL knock-out 2
8
1 Kb molecular weight marker
9
gRNA Ga20ox 1
10
gRNA Ga20ox 2
11
Ga20ox consensus 1
12
Ga20ox consensus 2
13
Ga20ox knock-out 1
14
Ga20ox knock-out 2
15
1 Kb molecular weight marker
Ligation using Golden Braid assembly of the next devises. Is used the restriction enzyme BsmbI.
Ordered the necessary primers to sequence: TFL consensus, TFL knockout, Ga20ox consensus and Ga20ox knockout.
E. coli transformations with:
Promoter 35s:Cas 9:Tnos – U6-26:TFL sgRNA: psgRNA
Plating the last devise in plates with Agrobacterium C58. Plates contain spec + IPTG + X-Gal. Incubate 2 days at 28°C.
Pick a single E. coli DH5α (promoter 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA and promoter 35s:Cas 9:Tnos – U6-26:Ga20ox sgRNA:psgRNA) colony from the plates that has been incubated overnight. Inoculate a starter culture of 4 ml of LB medium with 4 μL of spectinomycin in a 50 ml tube with the colony and incubate it overnight at 37°C with shaking.
23/07/2016
Minipreps (4 samples for each devise) with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
Digestion of minipreps with BamHI following digestion protocol . Incubate 1 hour at 37°C.
Run an electrophoresis gel of the digestion products. We will keep the minipreps with the sample number 4 for TFL sgRNA and the sample number 2 for Ga20ox sgRNA.
Pick a single Agrobacterium C58 (promoter 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA and promoter 35s:Cas 9:Tnos – U6-26:Ga20ox sgRNA:psgRNA ) colony from the plates that has been incubated overnight. Inoculate a starter culture of 5 ml of LB medium with 5 μL of spectinomycin and kanamycin in a falcon tube with the colony and incubate it overnight at 28°C with shaking.
Pick a single Agrobacterium C58 (promoter 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA in 3Ω1 and promoter 35s:Cas 9:Tnos – U6-26:Ga20ox sgRNA:psgRNA in 3Ω1 ) colony from the plates that has been incubated overnight. Inoculate a starter culture of 5 ml of LB medium with 5 μL of spectinomycin and kanamycin in a falcon tube with the colony and incubate it overnight at 28°C with shaking.
Incubate it 48 hours at 28°C
Minipreps of Agrobacterium with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
promoter 35s:5’ region:TFL Clemenules target control positive:Luc:Tnos
promoter 35s:5’ region:Ga20ox Gleva target control positive:Luc:Tnos
