Team:Valencia UPV/Notebook

• HOME
• TEAM
  • MEET US
  • COLLABORATIONS
  • ATTRIBUTIONS
• PROJECT
  • Description
  • DATA PROCESSING SOFTWARE
  • SPLIT Cas9
  • RESULTS
  • NOTEBOOK
• LAB WARE
  • HARDWARE
  • Reagents
  • Manual
• HUMAN PRACTICES
  • OVERVIEW
  • SOCIAL ENGAGEMENT
  • SOLVING REAL PROBLEMS
  • IMPACT ASSESSMENT
  • SAFETY
• PARTS
  • BEST PART COLLECTION
• MODEL
  • OVERVIEW
  • Cas9:gRNA Complex Formation
  • R-Loop Formation
  • Cleavage and Reading Frame Shift
  • Conclusions
• ACHIEVEMENTS

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°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:

Plasmid	GB Code
Promoter 35s:Cas9:nopaline synthase terminator (Tnos)	GB0639
Luciferase (Luc) in pUPD2	GB0096
Tnos in pUPD2	GB0037

01/07/2016

• Miniprep with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
• Promoter 35s:Cas9 : Tnos

• 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 in pUPD2
• Tnos in pUPD2

• Check orange DNA genome concentration with NanoDrop.

Sample	DNA concentration (ng / μL)
Clemenules1	3153.8
Clemenules 2	4527.9

• Perform a PCR to bind linker with luciferase:

Reagent	Volume(μL)	Program
LuciferasepUPD	1	Temperature	Time
Buffer HF	10	98°C	5 minutes
dNTPs	2	98°C	35x	30 seconds
IG16JUN01	2.5	70°C	35x	30 seconds
IG16JUN02	2.5	72°C	35x	1 minute 30 seconds
Taq phusion	0.5	72°C	10 minutes
H2O milli-Q	31.5	16°C	∞

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

• 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 Reaction

• 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 from Goldenbraid Collection:

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 in pUPD2
• U6-26 in pUPD2

• 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 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	TEMPERATURE	TIME
Buffer HF	10	98°C	5 minutes
dNTPs	2	98°C	35x	30 seconds
IG16JUL01 (TFL_For)	2.5	64°C	35x	30 seconds
IG16JUL02 (TFL_Rev)	2.5	72°C	35x	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	35x	30 seconds
IG16JUL02 (Ga20_rev)	2.5	72°C	35x	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.

• Primers IG16JUL09, IG16JUL10, IG16JUL11, IG16JUL12, IG16JUL13, IG16JUL14, IG16JUL15 and IG16JUL16 arrived.

• Ligation using Golden Braid assembly of the next devises:
• promoter 35s:5’ region:Clemenules target control positive:Luc:Tnos
• promoter 35s:5’ region:Gleva target control positive: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 1uM. It is necessary taking to 10 uM.

• 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 plasmid	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 plasmid	1
	BSA10X	1.2
	Ligase Buffer	1.2
	BsaI	1
	T4 ligase	1
	H2O milli-Q	3.6

• E. coli Transformation with the devises previously explained.

• E. coli plating in 6 plates (6 ligation reactions) with Kanamycin (antibiotic). Incubate overnight at 37°C.

• 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 37°C

15/07/2016

• Ligation using Golden Braid assembly of the next devises:
• Promoter 35s:Cas9:Tnos – promoter 35s:5’ region:TFL Clemenules target:Luc:Tnos
• Promoter 35s:Cas9:Tnos – promoter 35s:5’ region:Ga20ox Rice target:Luc:Tnos

• E. coli Transformation with the devises previously explained.

• Run an electrophoresis gel of the products from the digestion of minipreps. The devises are:
• Promoter 35s:5’ region:TFL Clemenules target:Luc:Tnos (3α1)
• Promoter 35s:5’ region :Ga20ox Rice target:Luc:Tnos (3α1)

• Sequencing the following products:
• Promotor 35s:5’ region in pUPD2 → correct

16/07/2016

• Transformations in E. coli DH5α with:
• Promoter 35s:5’ region:TFL Clemenules target:Luc:Tnos (3α1)
• Promoter 35s:5’ region:Ga20ox Rice target:Luc:Tnos (3α1)

• Plating the last devises in 2 plates with Agrobacterium C58. Incubate 2 days at 28°C.

• Minipreps (2 samples for each devise) with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
• Promoter 35s:5’ region:Clemenules target control positive:Luc:Tnos
• Promoter 35s:5’ region:Gleva target control positive: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 devises:
• Promotor 35s:Cas9:Tnos – U6:TFL Clemenules sgRNA:psgRNA (scaffold) (Ω1)
• Promotor 35s:Cas9:Tnos – U6: Ga20ox Rice sgRNA:psgRNA (scaffold) (Ω1)

Reagent	Volume (μL)
Promotor 35s:Cas9 : Tnos	1
U6-26:TFL Clemenules sgRNA:psgRNA / U6-26:Ga20ox rice 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 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	correct
2	gRNA Ga20ox 1	correct
3	gRNA Ga20ox 2	correct
4	Ga20ox consensus 1	correct
5	Ga20ox consensus 2	correct
6	Ga20ox knock-out 1	-
7	Ga20ox knock-out 2	correct
8	TFL consensus 1	-
9	TFL consensus 2	-
10	TFL knock-out 1	correct
11	TFL knock-out 2	correct
12	TFL gRNA 1	correct
13	TFL gRNA 2	correct
14	1 Kb molecular weight marker	correct

• 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.

17/07/2016

• Transformation in DH5α E. coli with:
• Promoter 35s:Cas9:Tnos – U6:TFL Clemenules sgRNA:psgRNA (scaffold) (Ω1)
• Promoter 35s:Cas9:Tnos – U6:Ga20ox sgRNA:psgRNA (scaffold) (Ω1)

• Plating E. coli transformations in plates with LB + agar + IPTG + XGal and incubate it overnight at 37°C.

• Miniprep with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
• Promoter U6-26:Ga20ox 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 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	correct
3	gRNA Ga20ox 2	correct
4	gRNA Ga20ox 3	correct
5	gRNA Ga20ox 4	correct
6	TFL knock-out 1	correct
7	TFL knock-out 2	correct
8	1 Kb molecular weight marker	correct

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
• Promoter 35s:5’ region:TFL Clemenules target consensus:Luc:Tnos
• Promoter 35s:5’ region:Ga20ox 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	promoter 35s: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	3α1	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

• Transformation in DH5α E. coli 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 37°C.

• Pick a single Agrobacterium C58 colony from the plates that have been incubating since 16/06/2016. The devises are:
• Promoter 35s:5’ region:TFL Clemenules target:Luc:Tnos (3α1)
• Promoter 35s:5’ region:Ga20ox Rice target:Luc:Tnos (3α1)

• Incubate it 48 hours at 28°C.

19/07/2016

• Pick transformed E. coli colony from the incubated plates. The devises are:
• promoter 35s:5’ region:TFL Clemenules target control positive:Luc:Tnos
• promoter 35s:5’ region:Ga20ox Gleva target control positive:Luc:Tnos
• promoter 35s:5’ region:TFL Clemenules target consensus:Luc:Tnos
• promoter 35s:5’ region:Ga20ox Gleva target consensus:Luc:Tnos
• promoter U6-26: sgRNA Clemenules:psgRNA (scaffold)
• promoter U6-26: sgRNA Gleva:psgRNA (scaffold)

• Incubate it overnight at 37°C.

20/07/2016

• Miniprep with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
• promoter 35s:5’ region:TFL Clemenules target knock-out:Luc:Tnos
• promoter 35s:5’ region:Ga20ox Gleva target control knock-out:Luc:Tnos
• promoter 35s:5’ region:TFL Clemenules target consensus:Luc:Tnos
• promoter 35s:5’ region:Ga20ox Gleva target consensus:Luc:Tnos
• promoter U6-26:sgRNA TFL Clemenules:psgRNA (scaffold)
• promoter U6-26:sgRNA Ga20ox Gleva:psgRNA (scaffold)

• 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	correct
2	gRNA TFL 1	correct
3	gRNA TFL 2	correct
4	TFL consensus 1	correct
5	TFL consensus 2	correct
6	TFL knock-out 1	correct
7	TFL knock-out 2	correct
8	1 Kb molecular weight marker	correct
9	gRNA Ga20ox 1	correct
10	gRNA Ga20ox 2	correct
11	Ga20ox consensus 1	correct
12	Ga20ox consensus 2	-
13	Ga20ox knock-out 1	correct
14	Ga20ox knock-out 2	correct
15	1 Kb molecular weight marker	correct

• Ligation using Golden Braid assembly of the next devises. Is used the restriction enzyme BsmbI.
• promoter 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA
• Promoter 35s:Cas 9:Tnos – U6-26:Ga20ox sgRNA:psgRNA

21/07/2016

• Transformations in E. coli DH5α with:
• promoter 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA
• promoter 35s:Cas 9:Tnos – U6-26:Ga20ox sgRNA:psgRNA

Incubate 2 hours at 37°C.

• Plating E. coli transformation explained before and incubate it at 37°C overnight.

• Agrobacterium C58 transformation with:
• promoter 35s:5’ region:TFL Clemenules target knock-out:Luc:Tnos
• promoter 35s:5’ region:Ga20ox Gleva target control knock-out:Luc:Tnos
• promoter 35s:5’ region:TFL Clemenules target consensus:Luc:Tnos
• promoter 35s:5’ region:Ga20ox Gleva target consensus:Luc:Tnos

Incubate 48 hours at 28°C.

22/07/2016

• Sequencing reaction:

Reagent	Volume (μL)
Primer in order to sequence	3
Miniprep reaction	5
H2O milli-Q	6

Sequence	Ordere
TFL gRNA	210.13.201
Ga20 gRNA	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: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:
• promoter 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA
• promoter 35s:Cas 9:Tnos – U6-26:Ga20ox sgRNA:psgRNA

• 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.

• Agrobacterium C58 transformations with:
• promoter 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA
• promoter 35s:Cas 9:Tnos – U6-26:Ga20ox sgRNA:psgRNA

24/07/2016

• Store the next cultures at -80°C:
• promoter 35s:5’ region in pUPD2 (DH5α) number 1
• Linker: luciferase in pUPD2 (DH5α) number 2

25/07/2016

• 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
• promoter 35s:5’ region:TFL Clemenules target consensus:Luc:Tnos
• promoter 35s:5’ region:Ga20ox Gleva target consensus:Luc:Tnos

• Digestion of minipreps with the restriction enzyme EcoRI. Incubate 1 hour at 37°C.

• Run an electrophoresis gel of the digestion products.

Lanes	Samples	Verification
1	1 Kb molecular weight marker	correct
2	Target Ga20ox consensus	correct
3	Target Ga20ox Knock-out	correct
4	Target TFL consensus	correct
5	Target TFL knock-out	correct
6	1 Kb molecular weight marker	correct

26/07/2016

• Minipreps with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
• promoter 35s:5’ region:TFL Clemenules target:Luc:Tnos in 3α1 plasmid from C58 Agrobacterium
• promoter 35s:5’ region:Ga20ox Gleva target:Luc:Tnos in 3α1 plasmid from C58 Agrobacterium

• Digestion of minipreps with the restriction enzyme EcoRI. Incubate 1 hour at 37°C.

• Run an electrophoresis gel of the digestion products:

Lanes	Samples	Verification
1	1 Kb molecular weight marker
2	promoter 35s:5’ region:Target Ga20ox Rice:Luc:Tnos
3	promoter 35s:5’ region:Target TFL Clemunules:Luc:Tnos
6	1 Kb molecular weight marker