Difference between revisions of "Team:Valencia UPV/Notebook"

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+
18/05/2016
 +
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 ºC.  
 +
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. (ENLACE)
 +
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 ºC overnight.
 +
Experiment with snails:
 +
Two experiments: one with infiltrated Nicotiana benthamiana and another with not infiltrated N.benthamiana (negative control). Lettuce leafs haven’t been correctly infiltrated and it seems that the expression level is low.
 +
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 haven’t been able to see fluorescence.
 +
30/06/2016
 +
Orange DNA Genome Extraction protocol (ENLACE)
 +
Take  from the glycerinates of GoldenBraid Collection:
 +
Plasmid
 +
GB Code
 +
Promoter 35S : Cas9 : nopaline synthase terminator (T-nos)
 +
GB0639
 +
Luciferase (Luc)
 +
GB0096
 +
T-nos
 +
GB0037
 +
 +
 +
01/07/2016
 +
Miniprep with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of:
 +
Promoter 35S : Cas9 : T-nos
 +
 +
Luciferase and nopaline synthase terminator cultures haven’t succeed. Repeat Luc and Tnos cultures.
 +
 +
Primers IG16JUN01 and IG16JUN02 have arrived.
 +
 +
Finish orange DNA Genome Extraction and check DNA concentration with NanoDrop. Concentration is very low so extraction will be done again.
 +
02/07/2016
 +
Miniprep with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of Luc and Tnos.
 +
 +
Check orange DNA genome concentration with NanoDrop.
 +
Sample
 +
DNA concentration (ng / μL)
 +
Clemenules 1
 +
3153.8
 +
Clemenules 2
 +
4527.9
 +
 +
Perform a PCR to bind linker with luciferase
 +
Reagent
 +
Volume (μL)
 +
Program
 +
Luciferase pUPD
 +
1
 +
Temperature
 +
Time
 +
Buffer HF
 +
10
 +
98 ºC
 +
5 minutes
 +
dNTPs
 +
2
 +
98 ºC
 +
35x
 +
30 seconds
 +
IG16JUN01
 +
2.5
 +
70 ºC
 +
30 seconds
 +
IG16JUN02
 +
2.5
 +
72 ºC
 +
1 minute 30 seconds
 +
Taq phusion
 +
0.5
 +
72 ºC
 +
10 minutes
 +
H2O milli-Q
 +
31.5
 +
16 ºC
 +
∞
 +
 +
04/07/2016
 +
Run electrophoresis gel of Clemenules DNA (agarose 1 %). 45 mL agarose gel at 1 % 0.45 g of agarose with 45 mL of TAE 1X. Voltage used is 100 V.
 +
 +
 +
Rice DNA Genome Extraction Protocol (ENLACE)
 +
 +
Run electrophoresis gel of Luciferase PCR product. 45 mL agarose gel at 1 % 0.45 g of agarose with 45 mL of TAE 1X. Voltage used is 100 V.
 +
 +
Ligate linker: Luciferase into a pUPD2 and transform E.coli DH5α with it. The method that is necessary to carry out this procedure is explained in protocols.
 +
 +
Check DNA concentration with NanoDrop.
 +
SAMPLE
 +
DNA Concentration (ng / μL)
 +
Rice Gleva 1
 +
22.8
 +
Rice Gleva 2
 +
17.3
 +
 +
05/07/2016
 +
 +
Run electrophoresis gel of Gleva rice DNA. We have checked that there isn’t DNA.
 +
 +
Repeat: Rice DNA Genome Extraction
 +
 +
Check DNA concentration with NanoDrop.
 +
SAMPLE
 +
DNA Concentration (ng / μL)
 +
Rice Gleva 1
 +
294.9
 +
Rice Gleva 2
 +
193.7
 +
 +
Run electrophoresis gel of Gleva rice DNA. It observed that genome extraction is correctly done.
 +
06/07/2016
 +
Take glycerinated cultures:
 +
GB part
 +
Plasmid
 +
Antibiotic
 +
Number GB
 +
psgRNA
 +
pUPD
 +
Ampicillin
 +
0645
 +
U6-26
 +
pUPD
 +
Ampicillin
 +
1001
 +
 +
07/07/2016
 +
Miniprep with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of psgRNA and U6-26
 +
 +
Primers IG16JUL01, IG16JUL02, IG16JUL03, IG16JUL04, IG16JUL05, IG16JUL06, IG16JUL07 and IG16JUL08 arrived.
 +
 +
gBlocks of - promoter 35S : 5’ region - have arrived.
 +
 +
We perform a PCR of orange and rice Genome Extraction.
 +
 +
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
 +
TEMPERATURE
 +
TIME
 +
Buffer HF
 +
10
 +
98ºC
 +
5 minutes
 +
dNTPs
 +
2
 +
98ºC
 +
35x
 +
30 seconds
 +
IG16JUL01 (TFL_For)
 +
2.5
 +
64ºC
 +
30 seconds
 +
IG16JUL02 (TFL_Rev)
 +
2.5
 +
72ºC
 +
30 seconds
 +
Taq phusion
 +
0.5
 +
72ºC
 +
10 minutes
 +
H2O milli-Q
 +
31.5
 +
16ºC
 +
∞
 +
 +
REAGENT
 +
VOLUME (μL)
 +
PROGRAM
 +
Gleva DNA
 +
1
 +
TEMPERATURE
 +
TIME
 +
Buffer HF
 +
10
 +
98ºC
 +
5 minutes
 +
dNTPs
 +
2
 +
98ºC
 +
35x
 +
30 seconds
 +
IG16JUL03 (Ga20_for)
 +
2.5
 +
72ºC
 +
30 seconds
 +
IG16JUL02 (Ga20_rev)
 +
2.5
 +
72ºC
 +
30 seconds
 +
Taq phusion
 +
0.5
 +
72ºC
 +
10 minutes
 +
H2O milli-Q
 +
31.5
 +
16ºC
 +
∞
 +
 +
 +
Ligate 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 construction: promoter 35S : 5’ region (electroporation 2.5KV). Plating it.
 +
 +
Take glycerinated culture for Georgia collaboration. The construction 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 35S : 5’ region petri dishes. Repeat transformation procedure and plating again.
 +
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 37ºC 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
 +
 +
Targets ligations ( 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 37ºC.
 +
 +
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 37ºC
 +
 +
Run electrophoresis gel of the construction: 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 37ºC.
 +
 +
Run electrophoresis gel of the same construction:
 +
Rice Gleva target in pUPD2
 +
Orange Clemenules target in pUPD2
 +
 +
Ligation using Golden Braid assembly of the next construction:
 +
Promoter 35S : 5’ region : Target : LUC : Tnos in α1
 +
13/07/2016
 +
E.coli Transformation with the construction:
 +
Promoter 35S : 5’ region : Target : LUC : Tnos in α1
 +
 +
E. coli plating in plates with Kanamycin (antibiotic). Incubate overnight at 37ºC
 +
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 37ºC.
 +
 +
Primers IG16JUL09, IG16JUL10, IG16JUL11, IG16JUL12, IG16JUL13, IG16JUL14, IG16JUL15 and IG16JUL16 arrived.
 +
 +
Ligation using Golden Braid assembly of the next constructions:
 +
Promoter 35S : 5’Region : Clemenules target control + : Luc : Tnos
 +
Promoter 35S : 5’Region : Gleva target control + : Luc : Tnos
 +
Promoter 35S : 5’Region : Clemenules target consensus : Luc : Tnos
 +
Promoter 35S : 5’Region : Gleva target consensus : Luc : Tnos
 +
Promoter U6-26 : sgRNA Clemenules : psgRNA (scaffold)
 +
Promoter U6-26 : sgRNA Gleva : psgRNA (scaffold)
 +
 +
Step 1: Annealing of oligonucleotides. Received primers are at 1μM. It is necessary taking to 10 μM.
 +
Mix in an Eppendorf:
 +
18 μL of H2O milli-Q
 +
1 μL forward primer
 +
1 μL reverse primer
 +
Step 2: Ligation reaction
 +
Reagent
 +
Volume (μL)
 +
Clemenules target control + / Gleva target control + / Clemenules target consensus / Gleva target consensus
 +
1
 +
Promoter 35S : 5’ region
 +
1
 +
Luciferase
 +
1
 +
Tnos
 +
1
 +
α1
 +
1
 +
BSA10X
 +
1.2
 +
Ligase Buffer
 +
1.2
 +
BsaI
 +
1
 +
T4 ligase
 +
1
 +
H2O milli-Q
 +
2.6
 +
 +
Reagent
 +
Volume (μL)
 +
sgRNA Clemenules / sgRNA Gleva
 +
1
 +
Promoter U6 -26
 +
1
 +
psgRNA (scaffold)
 +
1
 +
α1
 +
1
 +
BSA10X
 +
1.2
 +
Ligase Buffer
 +
1.2
 +
BsaI
 +
1
 +
T4 ligase
 +
1
 +
H2O milli-Q
 +
3.6
 +
 +
E.coli Transformation with the constructions previously explained.
 +
 +
E. coli plating in 6 plates (6 ligation reactions) with Kanamycin (antibiotic). Incubate overnight at 37ºC.
 +
 +
Miniprep with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of:
 +
Promoter 35S : 5’ region : CL target : Luc : Tnos (3α1)
 +
Promoter 35S : 5’ region : A target : Luc : Tnos (3α1)
 +
 +
Digestion of minipreps with EcoRI. Incubate 1 hour at 37ºC
 +
15/07/2016
 +
Ligation using Golden Braid assembly of the next construction:
 +
Promotor 35S : Cas9 : Tnos – Promotor 35S : 5’ region : CL target : Luc : Tnos
 +
Promotor 35S : Cas9 : Tnos – promotor 35S : 5’ region : A target : Luc : Tnos
 +
 +
E. coli Transformation with the constructions previously explained.
 +
 +
Run electrophoresis gel of the products from the digestion of minipreps. The constructions are:
 +
Promoter 35S : 5’ region : CL target : Luc : Tnos (3α1)
 +
Promoter 35S : 5’ region : A target : Luc : Tnos (3α1)
 +
 +
Sequencing products:
 +
Promotor 35S : 5’ region in pUPD2 → correct
 +
16/07/2016
 +
 +
E.coli transformations with:
 +
Promoter 35S : 5’ region : CL target : Luc : Tnos (3α1)
 +
Promoter 35S : 5’ region : A target : Luc : Tnos (3α1)
 +
 +
Plating the last constructions in 2 plates with Agrobacterium C58. Incubate 2 days at 28ºC.
 +
 +
Minipreps (2 samples for each construction) with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of:
 +
Promoter 35S : 5’Region : Clemenules target control + : Luc : Tnos
 +
Promoter 35S : 5’Region : Gleva target control + : Luc : Tnos
 +
Promoter 35S : 5’Region : Clemenules target consensus : Luc : Tnos
 +
Promoter 35S : 5’Region : Gleva target consensus : Luc : Tnos
 +
Promoter U6-26 : sgRNA Clemenules : psgRNA (scaffold)
 +
Promoter U6-26 : sgRNA Gleva : psgRNA (scaffold)
 +
 +
Ligation using Golden Braid assembly of the next construction:
 +
Promotor 35S : Cas9 : Tnos –U6 : CL sgRNA : psgRNA (scaffold) (Ω1)
 +
Promotor 35S : Cas9 : Tnos –U6 : A sgRNA : psgRNA (scaffold) (Ω1)
 +
Reagent
 +
Volume (μL)
 +
Promotor 35S : Cas9 : Tnos
 +
1
 +
U6-26 : CL sgRNA : psgRNA /                  U6-26 : A sgRNA : psgRNA
 +
1
 +
3Ω1
 +
1
 +
BSA10X
 +
1.2
 +
Ligase Buffer
 +
1.2
 +
BsmbI
 +
1
 +
T4 ligase
 +
1
 +
H2O milli-Q
 +
4.6
 +
 +
Digestion of the minipreps with EcoRI which have been done before. Incubate 1 hour at 37ºC. 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 constructions are:
 +
Promotor 35S : TFL Knock-out : Luc : Tnos (4 samples)
 +
Promoter U6-26 : A sgRNA : psgRNA (2 samples)
 +
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 37ºC.
 +
 +
17/07/2016
 +
DH5α E.coli transformations with:
 +
Promotor 35S : Cas9 : Tnos –U6 : CL sgRNA : psgRNA (scaffold) (Ω1)
 +
Promotor 35S : Cas9 : Tnos –U6 : A sgRNA : psgRNA (scaffold) (Ω1)
 +
 +
Plating E.coli transformations in plates with LB + agar + IPTG + XGal and incubate it overnight at 37ºC.
 +
 +
Miniprep with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of:
 +
Promoter U6-26 : A sgRNA : psgRNA
 +
Promoter 35S : 5’ region : TFL Knock-out : Luc : Tnos (3α1)
 +
 +
Digestion of the minipreps with EcoRI which have been done before. Incubate 1 hour at 37ºC. There is a total of six 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
 +
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 haven’t get the expected results for a gRNA.
 +
 +
Ligation using Golden Braid assembly of the next constructions:
 +
Promoter 35S : 5’Region : Clemenules target control + : Luc : Tnos
 +
Promoter 35S : 5’Region : Gleva target control + : Luc : Tnos
 +
Promoter 35S : 5’Region : Clemenules target consensus : Luc : Tnos
 +
Promoter 35S : 5’Region : Gleva target consensus : Luc : Tnos
 +
Promoter U6-26 : sgRNA Clemenules : psgRNA (scaffold)
 +
Promoter U6-26 : sgRNA Gleva : psgRNA (scaffold)
 +
Reagent
 +
Volume (μL)
 +
Reagent
 +
Volume (μL)
 +
TFL/Ga20ox gRNA
 +
1
 +
Promotor35S :5’region
 +
1
 +
U6-26
 +
1
 +
TFL / Ga20ox consensus          TFL / Ga20ox knock-out
 +
1
 +
psgRNA
 +
1
 +
luciferase
 +
1
 +
3α1
 +
1
 +
Tnos
 +
1
 +
BSA10X
 +
1.2
 +
3Ω1
 +
1
 +
Ligase Buffer
 +
1.2
 +
BSA10X
 +
1.2
 +
BsaI
 +
1
 +
Ligase Buffer
 +
1.2
 +
T4 ligase
 +
1
 +
BsmbI
 +
1
 +
H2O milli-Q
 +
3.6
 +
T4 ligase
 +
1
 +
 +
 +
 +
 +
H2O milli-Q
 +
2.6
 +
 +
18/07/2016
 +
DH5α E.coli transformations with ligation products (consensus targets and knock-out targets of TFL and Ga20ox as well as their gRNAs)
 +
 +
Plating E.coli transformations in plates with LB + agar + IPTG + XGal and incubate it overnight at 37ºC.
 +
 +
Pick a single Agrobacterium C58 colony from the plates that have been incubating since 16/06/2016. The constructions are:
 +
Promoter 35S : 5’ region : CL target : Luc : Tnos (3α1)
 +
Promoter 35S : 5’ region : A target : Luc : Tnos (3α1)
 +
Incubate it 48 hours at 28ºC.
 +
19/07/2016
 +
 +
Pick transformed E.coli colony from the incubated plates. The constructions are:
 +
Promoter 35S : 5’Region : Clemenules target control + : Luc : Tnos
 +
Promoter 35S : 5’Region : Gleva target control + : Luc : Tnos
 +
Promoter 35S : 5’Region : Clemenules target consensus : Luc : Tnos
 +
Promoter 35S : 5’Region : Gleva target consensus : Luc : Tnos
 +
Promoter U6-26 : sgRNA Clemenules : psgRNA (scaffold)
 +
Promoter U6-26 : sgRNA Gleva : psgRNA (scaffold)
 +
    Incubate it overnight at 37ºC.
 +
20/07/2016
 +
 +
-          Miniprep with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of:
 +
§  Promoter 35S : 5’Region : Clemenules target knockout : Luc : Tnos
 +
§  Promoter 35S : 5’Region : Gleva target control knockout : Luc : Tnos
 +
§  Promoter 35S : 5’Region : Clemenules target consensus : Luc : Tnos
 +
§  Promoter 35S : 5’Region : Gleva target consensus : Luc : Tnos
 +
§  Promoter U6-26 : sgRNA Clemenules : psgRNA (scaffold)
 +
§  Promoter U6-26 : sgRNA Gleva : psgRNA (scaffold)
 +
 +
-          Digestion of the minipreps with EcoRI which have been done before. Incubate 1 hour at 37ºC. There is a total of six minipreps. It has been followed the Miniprep digestion protocol.
 +
 +
-          Run 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 constructions. Is used the restriction enzyme BsmbI.
 +
§  Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA
 +
§  Promoter 35s:Cas 9:T-nos – U6-26:Ga20ox sgRNA: psgRNA
 +
21/07/2016
 +
 +
-          E.coli transformations with:
 +
§  Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA
 +
§  Promoter 35s:Cas 9:T-nos – U6-26:Ga20ox sgRNA: psgRNA
 +
Incubate 2 hours at 37 ºC.
 +
-          Plating E.coli transformation explained before and incubate it at 37ºC overnight.
 +
 +
-          Agrobacterium C58 transformation with:
 +
§  Promoter 35S : 5’Region : Clemenules target knockout : Luc : Tnos
 +
§  Promoter 35S : 5’Region : Gleva target control knockout : Luc : Tnos
 +
§  Promoter 35S : 5’Region : Clemenules target consensus : Luc : Tnos
 +
§  Promoter 35S : 5’Region : Gleva target consensus : Luc : Tnos
 +
Incubate 48 hours at 28 ºC.
 +
 +
22/07/2016
 +
 +
-          Sequencing reaction
 +
Reagent
 +
Volume (μL)
 +
Primer to sequence
 +
3
 +
Miniprep reaction
 +
5
 +
H2O milli-Q
 +
6
 +
 +
TFL gRNA à 210.13.201
 +
Ga20 gRNA à 210.13.202
 +
-          Ordered the necessary primers to sequence:
 +
TFL consensus, TFL knockout, Ga20ox consensus and Ga20ox knockout.
 +
-          E.coli transformations with:
 +
§  Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA
 +
 +
-          Plating the last construction in plates with Agrobacterium C58. Plates contain spec + IPTG + X-Gal. Incubate 2 days at 28ºC.
 +
 +
-          Pick a single E.coli DH5α (Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA and Promoter 35s:Cas 9:T-nos – 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 37ºC with shaking.
 +
 +
23/07/2016
 +
 +
-          Minipreps (4 samples for each construction) with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of:
 +
§  Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA
 +
§  Promoter 35s:Cas 9:T-nos – U6-26:Ga20ox sgRNA: psgRNA
 +
 +
-          Digestion of minipreps with BamHI following digestion protocol. Incubate 1 hour at 37ºC.
 +
 +
-          Run 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:T-nos – U6-26:TFL sgRNA: psgRNA and Promoter 35s:Cas 9:T-nos – 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 28ºC with shaking.
 +
 +
-          Agrobacterium C58 transformations with:
 +
§  Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA
 +
§  Promoter 35s:Cas 9:T-nos – U6-26:Ga20ox sgRNA: psgRNA
 +
24/07/2016
 +
 +
-          Store the next cultures at -80 ºC:
 +
§  Promoter 35s:5’ region in pUPD2 (DH5α) à 1
 +
§  Linker: luciferase in pUPD2 (DH5α) à 2
 +
25/07/2016
 +
 +
-          Pick a single Agrobacterium C58 (Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA in 3Ω1 and promoter 35s:Cas 9:T-nos – 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 28ºC with shaking.
 +
 +
Incubate it 48 hours at 28 ºC
 +
 +
-          Minipreps of Agrobacterium with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of:
 +
§  Promoter 35S : 5’Region : Clemenules target control + : Luc : Tnos
 +
§  Promoter 35S : 5’Region : Gleva target control + : Luc : Tnos
 +
§  Promoter 35S : 5’Region : Clemenules target consensus : Luc : Tnos
 +
§  Promoter 35S : 5’Region : Gleva target consensus : Luc : Tnos
 +
 +
-          Digestion of minipreps with the restriction enzyme EcoRI. Incubate 1 hour at 37ºC.
 +
 +
-          Run electrophoresis gel of the digestion products.
 +
 +
Lanes
 +
Samples
 +
Verification
 +
1
 +
1 Kb molecular weight marker
 +
 +
 +
2
 +
Target Ga20ox consensus
 +
 +
 +
3
 +
Target Ga20ox Knockout
 +
 +
 +
4
 +
Target TFL consensus
 +
 +
 +
5
 +
Target TFL knockout
 +
 +
 +
6
 +
1 Kb molecular weight marker
  
 
     </p>
 
     </p>

Revision as of 16:07, 25 July 2016

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18/05/2016 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 ºC. 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. (ENLACE) 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 ºC overnight. Experiment with snails: Two experiments: one with infiltrated Nicotiana benthamiana and another with not infiltrated N.benthamiana (negative control). Lettuce leafs haven’t been correctly infiltrated and it seems that the expression level is low. 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 haven’t been able to see fluorescence. 30/06/2016 Orange DNA Genome Extraction protocol (ENLACE) Take from the glycerinates of GoldenBraid Collection: Plasmid GB Code Promoter 35S : Cas9 : nopaline synthase terminator (T-nos) GB0639 Luciferase (Luc) GB0096 T-nos GB0037 01/07/2016 Miniprep with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of: Promoter 35S : Cas9 : T-nos Luciferase and nopaline synthase terminator cultures haven’t succeed. Repeat Luc and Tnos cultures. Primers IG16JUN01 and IG16JUN02 have arrived. Finish orange DNA Genome Extraction and check DNA concentration with NanoDrop. Concentration is very low so extraction will be done again. 02/07/2016 Miniprep with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of Luc and Tnos. Check orange DNA genome concentration with NanoDrop. Sample DNA concentration (ng / μL) Clemenules 1 3153.8 Clemenules 2 4527.9 Perform a PCR to bind linker with luciferase Reagent Volume (μL) Program Luciferase pUPD 1 Temperature Time Buffer HF 10 98 ºC 5 minutes dNTPs 2 98 ºC 35x 30 seconds IG16JUN01 2.5 70 ºC 30 seconds IG16JUN02 2.5 72 ºC 1 minute 30 seconds Taq phusion 0.5 72 ºC 10 minutes H2O milli-Q 31.5 16 ºC ∞ 04/07/2016 Run electrophoresis gel of Clemenules DNA (agarose 1 %). 45 mL agarose gel at 1 % 0.45 g of agarose with 45 mL of TAE 1X. Voltage used is 100 V. Rice DNA Genome Extraction Protocol (ENLACE) Run electrophoresis gel of Luciferase PCR product. 45 mL agarose gel at 1 % 0.45 g of agarose with 45 mL of TAE 1X. Voltage used is 100 V. Ligate linker: Luciferase into a pUPD2 and transform E.coli DH5α with it. The method that is necessary to carry out this procedure is explained in protocols. Check DNA concentration with NanoDrop. SAMPLE DNA Concentration (ng / μL) Rice Gleva 1 22.8 Rice Gleva 2 17.3 05/07/2016 Run electrophoresis gel of Gleva rice DNA. We have checked that there isn’t DNA. Repeat: Rice DNA Genome Extraction Check DNA concentration with NanoDrop. SAMPLE DNA Concentration (ng / μL) Rice Gleva 1 294.9 Rice Gleva 2 193.7 Run electrophoresis gel of Gleva rice DNA. It observed that genome extraction is correctly done. 06/07/2016 Take glycerinated cultures: GB part Plasmid Antibiotic Number GB psgRNA pUPD Ampicillin 0645 U6-26 pUPD Ampicillin 1001 07/07/2016 Miniprep with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of psgRNA and U6-26 Primers IG16JUL01, IG16JUL02, IG16JUL03, IG16JUL04, IG16JUL05, IG16JUL06, IG16JUL07 and IG16JUL08 arrived. gBlocks of - promoter 35S : 5’ region - have arrived. We perform a PCR of orange and rice Genome Extraction. 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 TEMPERATURE TIME Buffer HF 10 98ºC 5 minutes dNTPs 2 98ºC 35x 30 seconds IG16JUL01 (TFL_For) 2.5 64ºC 30 seconds IG16JUL02 (TFL_Rev) 2.5 72ºC 30 seconds Taq phusion 0.5 72ºC 10 minutes H2O milli-Q 31.5 16ºC ∞ REAGENT VOLUME (μL) PROGRAM Gleva DNA 1 TEMPERATURE TIME Buffer HF 10 98ºC 5 minutes dNTPs 2 98ºC 35x 30 seconds IG16JUL03 (Ga20_for) 2.5 72ºC 30 seconds IG16JUL02 (Ga20_rev) 2.5 72ºC 30 seconds Taq phusion 0.5 72ºC 10 minutes H2O milli-Q 31.5 16ºC ∞ Ligate 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 construction: promoter 35S : 5’ region (electroporation 2.5KV). Plating it. Take glycerinated culture for Georgia collaboration. The construction 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 35S : 5’ region petri dishes. Repeat transformation procedure and plating again. 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 37ºC 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 Targets ligations ( 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 37ºC. 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 37ºC Run electrophoresis gel of the construction: 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 37ºC. Run electrophoresis gel of the same construction: Rice Gleva target in pUPD2 Orange Clemenules target in pUPD2 Ligation using Golden Braid assembly of the next construction: Promoter 35S : 5’ region : Target : LUC : Tnos in α1 13/07/2016 E.coli Transformation with the construction: Promoter 35S : 5’ region : Target : LUC : Tnos in α1 E. coli plating in plates with Kanamycin (antibiotic). Incubate overnight at 37ºC 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 37ºC. Primers IG16JUL09, IG16JUL10, IG16JUL11, IG16JUL12, IG16JUL13, IG16JUL14, IG16JUL15 and IG16JUL16 arrived. Ligation using Golden Braid assembly of the next constructions: Promoter 35S : 5’Region : Clemenules target control + : Luc : Tnos Promoter 35S : 5’Region : Gleva target control + : Luc : Tnos Promoter 35S : 5’Region : Clemenules target consensus : Luc : Tnos Promoter 35S : 5’Region : Gleva target consensus : Luc : Tnos Promoter U6-26 : sgRNA Clemenules : psgRNA (scaffold) Promoter U6-26 : sgRNA Gleva : psgRNA (scaffold) Step 1: Annealing of oligonucleotides. Received primers are at 1μM. It is necessary taking to 10 μM. Mix in an Eppendorf: 18 μL of H2O milli-Q 1 μL forward primer 1 μL reverse primer Step 2: Ligation reaction Reagent Volume (μL) Clemenules target control + / Gleva target control + / Clemenules target consensus / Gleva target consensus 1 Promoter 35S : 5’ region 1 Luciferase 1 Tnos 1 α1 1 BSA10X 1.2 Ligase Buffer 1.2 BsaI 1 T4 ligase 1 H2O milli-Q 2.6 Reagent Volume (μL) sgRNA Clemenules / sgRNA Gleva 1 Promoter U6 -26 1 psgRNA (scaffold) 1 α1 1 BSA10X 1.2 Ligase Buffer 1.2 BsaI 1 T4 ligase 1 H2O milli-Q 3.6 E.coli Transformation with the constructions previously explained. E. coli plating in 6 plates (6 ligation reactions) with Kanamycin (antibiotic). Incubate overnight at 37ºC. Miniprep with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of: Promoter 35S : 5’ region : CL target : Luc : Tnos (3α1) Promoter 35S : 5’ region : A target : Luc : Tnos (3α1) Digestion of minipreps with EcoRI. Incubate 1 hour at 37ºC 15/07/2016 Ligation using Golden Braid assembly of the next construction: Promotor 35S : Cas9 : Tnos – Promotor 35S : 5’ region : CL target : Luc : Tnos Promotor 35S : Cas9 : Tnos – promotor 35S : 5’ region : A target : Luc : Tnos E. coli Transformation with the constructions previously explained. Run electrophoresis gel of the products from the digestion of minipreps. The constructions are: Promoter 35S : 5’ region : CL target : Luc : Tnos (3α1) Promoter 35S : 5’ region : A target : Luc : Tnos (3α1) Sequencing products: Promotor 35S : 5’ region in pUPD2 → correct 16/07/2016 E.coli transformations with: Promoter 35S : 5’ region : CL target : Luc : Tnos (3α1) Promoter 35S : 5’ region : A target : Luc : Tnos (3α1) Plating the last constructions in 2 plates with Agrobacterium C58. Incubate 2 days at 28ºC. Minipreps (2 samples for each construction) with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of: Promoter 35S : 5’Region : Clemenules target control + : Luc : Tnos Promoter 35S : 5’Region : Gleva target control + : Luc : Tnos Promoter 35S : 5’Region : Clemenules target consensus : Luc : Tnos Promoter 35S : 5’Region : Gleva target consensus : Luc : Tnos Promoter U6-26 : sgRNA Clemenules : psgRNA (scaffold) Promoter U6-26 : sgRNA Gleva : psgRNA (scaffold) Ligation using Golden Braid assembly of the next construction: Promotor 35S : Cas9 : Tnos –U6 : CL sgRNA : psgRNA (scaffold) (Ω1) Promotor 35S : Cas9 : Tnos –U6 : A sgRNA : psgRNA (scaffold) (Ω1) Reagent Volume (μL) Promotor 35S : Cas9 : Tnos 1 U6-26 : CL sgRNA : psgRNA / U6-26 : A sgRNA : psgRNA 1 3Ω1 1 BSA10X 1.2 Ligase Buffer 1.2 BsmbI 1 T4 ligase 1 H2O milli-Q 4.6 Digestion of the minipreps with EcoRI which have been done before. Incubate 1 hour at 37ºC. 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 constructions are: Promotor 35S : TFL Knock-out : Luc : Tnos (4 samples) Promoter U6-26 : A sgRNA : psgRNA (2 samples) 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 37ºC. 17/07/2016 DH5α E.coli transformations with: Promotor 35S : Cas9 : Tnos –U6 : CL sgRNA : psgRNA (scaffold) (Ω1) Promotor 35S : Cas9 : Tnos –U6 : A sgRNA : psgRNA (scaffold) (Ω1) Plating E.coli transformations in plates with LB + agar + IPTG + XGal and incubate it overnight at 37ºC. Miniprep with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of: Promoter U6-26 : A sgRNA : psgRNA Promoter 35S : 5’ region : TFL Knock-out : Luc : Tnos (3α1) Digestion of the minipreps with EcoRI which have been done before. Incubate 1 hour at 37ºC. There is a total of six 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 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 haven’t get the expected results for a gRNA. Ligation using Golden Braid assembly of the next constructions: Promoter 35S : 5’Region : Clemenules target control + : Luc : Tnos Promoter 35S : 5’Region : Gleva target control + : Luc : Tnos Promoter 35S : 5’Region : Clemenules target consensus : Luc : Tnos Promoter 35S : 5’Region : Gleva target consensus : Luc : Tnos Promoter U6-26 : sgRNA Clemenules : psgRNA (scaffold) Promoter U6-26 : sgRNA Gleva : psgRNA (scaffold) Reagent Volume (μL) Reagent Volume (μL) TFL/Ga20ox gRNA 1 Promotor35S :5’region 1 U6-26 1 TFL / Ga20ox consensus TFL / Ga20ox knock-out 1 psgRNA 1 luciferase 1 3α1 1 Tnos 1 BSA10X 1.2 3Ω1 1 Ligase Buffer 1.2 BSA10X 1.2 BsaI 1 Ligase Buffer 1.2 T4 ligase 1 BsmbI 1 H2O milli-Q 3.6 T4 ligase 1 H2O milli-Q 2.6 18/07/2016 DH5α E.coli transformations with ligation products (consensus targets and knock-out targets of TFL and Ga20ox as well as their gRNAs) Plating E.coli transformations in plates with LB + agar + IPTG + XGal and incubate it overnight at 37ºC. Pick a single Agrobacterium C58 colony from the plates that have been incubating since 16/06/2016. The constructions are: Promoter 35S : 5’ region : CL target : Luc : Tnos (3α1) Promoter 35S : 5’ region : A target : Luc : Tnos (3α1) Incubate it 48 hours at 28ºC. 19/07/2016 Pick transformed E.coli colony from the incubated plates. The constructions are: Promoter 35S : 5’Region : Clemenules target control + : Luc : Tnos Promoter 35S : 5’Region : Gleva target control + : Luc : Tnos Promoter 35S : 5’Region : Clemenules target consensus : Luc : Tnos Promoter 35S : 5’Region : Gleva target consensus : Luc : Tnos Promoter U6-26 : sgRNA Clemenules : psgRNA (scaffold) Promoter U6-26 : sgRNA Gleva : psgRNA (scaffold) Incubate it overnight at 37ºC. 20/07/2016 - Miniprep with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of: § Promoter 35S : 5’Region : Clemenules target knockout : Luc : Tnos § Promoter 35S : 5’Region : Gleva target control knockout : Luc : Tnos § Promoter 35S : 5’Region : Clemenules target consensus : Luc : Tnos § Promoter 35S : 5’Region : Gleva target consensus : Luc : Tnos § Promoter U6-26 : sgRNA Clemenules : psgRNA (scaffold) § Promoter U6-26 : sgRNA Gleva : psgRNA (scaffold) - Digestion of the minipreps with EcoRI which have been done before. Incubate 1 hour at 37ºC. There is a total of six minipreps. It has been followed the Miniprep digestion protocol. - Run 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 constructions. Is used the restriction enzyme BsmbI. § Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA § Promoter 35s:Cas 9:T-nos – U6-26:Ga20ox sgRNA: psgRNA 21/07/2016 - E.coli transformations with: § Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA § Promoter 35s:Cas 9:T-nos – U6-26:Ga20ox sgRNA: psgRNA Incubate 2 hours at 37 ºC. - Plating E.coli transformation explained before and incubate it at 37ºC overnight. - Agrobacterium C58 transformation with: § Promoter 35S : 5’Region : Clemenules target knockout : Luc : Tnos § Promoter 35S : 5’Region : Gleva target control knockout : Luc : Tnos § Promoter 35S : 5’Region : Clemenules target consensus : Luc : Tnos § Promoter 35S : 5’Region : Gleva target consensus : Luc : Tnos Incubate 48 hours at 28 ºC. 22/07/2016 - Sequencing reaction Reagent Volume (μL) Primer to sequence 3 Miniprep reaction 5 H2O milli-Q 6 TFL gRNA à 210.13.201 Ga20 gRNA à 210.13.202 - Ordered the necessary primers to sequence: TFL consensus, TFL knockout, Ga20ox consensus and Ga20ox knockout. - E.coli transformations with: § Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA - Plating the last construction in plates with Agrobacterium C58. Plates contain spec + IPTG + X-Gal. Incubate 2 days at 28ºC. - Pick a single E.coli DH5α (Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA and Promoter 35s:Cas 9:T-nos – 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 37ºC with shaking. 23/07/2016 - Minipreps (4 samples for each construction) with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of: § Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA § Promoter 35s:Cas 9:T-nos – U6-26:Ga20ox sgRNA: psgRNA - Digestion of minipreps with BamHI following digestion protocol. Incubate 1 hour at 37ºC. - Run 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:T-nos – U6-26:TFL sgRNA: psgRNA and Promoter 35s:Cas 9:T-nos – 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 28ºC with shaking. - Agrobacterium C58 transformations with: § Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA § Promoter 35s:Cas 9:T-nos – U6-26:Ga20ox sgRNA: psgRNA 24/07/2016 - Store the next cultures at -80 ºC: § Promoter 35s:5’ region in pUPD2 (DH5α) à 1 § Linker: luciferase in pUPD2 (DH5α) à 2 25/07/2016 - Pick a single Agrobacterium C58 (Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA in 3Ω1 and promoter 35s:Cas 9:T-nos – 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 28ºC with shaking. Incubate it 48 hours at 28 ºC - Minipreps of Agrobacterium with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of: § Promoter 35S : 5’Region : Clemenules target control + : Luc : Tnos § Promoter 35S : 5’Region : Gleva target control + : Luc : Tnos § Promoter 35S : 5’Region : Clemenules target consensus : Luc : Tnos § Promoter 35S : 5’Region : Gleva target consensus : Luc : Tnos - Digestion of minipreps with the restriction enzyme EcoRI. Incubate 1 hour at 37ºC. - Run electrophoresis gel of the digestion products. Lanes Samples Verification 1 1 Kb molecular weight marker 2 Target Ga20ox consensus 3 Target Ga20ox Knockout 4 Target TFL consensus 5 Target TFL knockout 6 1 Kb molecular weight marker