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

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<tr><td>dNTPs</td><td>2</td><td>98&deg;C</td><td style="text-align: center; vertical-align: middle;" rowspan="3" >35x</td><td>30 seconds</td></tr>
 
<tr><td>dNTPs</td><td>2</td><td>98&deg;C</td><td style="text-align: center; vertical-align: middle;" rowspan="3" >35x</td><td>30 seconds</td></tr>
  
<tr><td>IG16JUN01</td><td>2.5</td><td>70&deg;C</td><td>35x</td><td>30 seconds</td></tr>
+
<tr><td>IG16JUN01</td><td>2.5</td><td>70&deg;C</td><td>30 seconds</td></tr>
  
<tr><td>IG16JUN02</td><td>2.5</td><td>72&deg;C</td><td>35x</td><td>1 minute 30 seconds</td></tr>
+
<tr><td>IG16JUN02</td><td>2.5</td><td>72&deg;C</td><td>1 minute 30 seconds</td></tr>
  
 
<tr><td>Taq phusion</td><td>0.5</td><td>72&deg;C</td><td>10 minutes</td></tr>
 
<tr><td>Taq phusion</td><td>0.5</td><td>72&deg;C</td><td>10 minutes</td></tr>

Revision as of 10:14, 26 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°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:
PlasmidGB Code
pD6B3 α1GB0015
pD6B3 α2GB0017
pD6B3 Ω1GB0019
pD6B3 Ω2GB0021
pUPD2GB0307
  • 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:
PlasmidGB Code
Promoter 35s:Cas9:nopaline synthase terminator (Tnos)GB0639
Luciferase (Luc) in pUPD2GB0096
Tnos in pUPD2GB0037

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.
SampleDNA concentration (ng / μL)
Clemenules1 3153.8
Clemenules 2 4527.9
  • Perform a PCR to bind linker with luciferase:
ReagentVolume(μL)Program
LuciferasepUPD1TemperatureTime
Buffer HF1098°C5 minutes
dNTPs298°C35x30 seconds
IG16JUN012.570°C30 seconds
IG16JUN022.572°C1 minute 30 seconds
Taq phusion0.572°C10 minutes
H2O milli-Q31.516°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.
SAMPLEDNA Concentration(ng / μL)
Rice Gleva 122.8
Rice Gleva 217.3

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.
SAMPLEDNA Concentration(ng / μL)
Rice Gleva 1294.9
Rice Gleva 2193.7
  • 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 partPlasmidAntibioticNumber GB
psgRNApUPDAmpicillin0645
U6-26pUPDAmpicillin1001

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
SampleInitial concentration(ng/μL)Final concentration(ng/μL)Initial volume(μL)Final volume (μL)
Clemenules 13153.81504.756100
Clemenules 24527.91503.31100
Gleva 1294.915050.8647100
Gleva 2193.715077.44100
REAGENTVOLUME(μL)PROGRAM
Clemenules DNA 1TEMPERATURETIME
Buffer HF1098°C5 minutes
dNTPs298°C35x30 seconds
IG16JUL01 (TFL_For)2.564°C35x30 seconds
IG16JUL02 (TFL_Rev)2.572°C35x30 seconds
Taq phusion0.572°C10 minutes
H2O milli-Q31.516°C
REAGENTVOLUME(μL)PROGRAM
Gleva DNA1TEMPERATURETIME
Buffer HF1098°C5 minutes
dNTPs298°C35x30 seconds
IG16JUL03 (Ga20_for)2.572°C35x30 seconds
IG16JUL02 (Ga20_rev)2.572°C35x30 seconds
Taq phusion0.572°C10 minutes
H2O milli-Q31.516°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)
SampleDNA Concentration(ng / μL)DNA Concentration(ng)
1105.25035.2
2104.65035.2
  • 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
ReagentVolume (μL)
Clemenules target control + / Gleva target control + / Clemenules target consensus / Gleva target consensus1
Promoter 35s:5’ region1
Luciferase1
Tnos1
α1 plasmid1
BSA10X1.2
Ligase Buffer1.2
BsaI1
T4 ligase1
H2O milli-Q2.6

ReagentVolume (μL)
sgRNA Clemenules / sgRNA Gleva1
Promoter U6 -261
psgRNA (scaffold)1
α1 plasmid1
BSA10X1.2
Ligase Buffer1.2
BsaI1
T4 ligase1
H2O milli-Q3.6

  • 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)
ReagentVolume (μL)
Promotor 35s:Cas9 : Tnos1
U6-26:TFL Clemenules sgRNA:psgRNA / U6-26:Ga20ox rice sgRNA:psgRNA1
3Ω11
BSA10X1.2
Ligase Buffer1.2
BsmbI1
T4 ligase1
H2O milli-Q4.6
  • 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.
LanesSamplesVerification
11 Kb molecular weight markercorrect
2gRNA Ga20ox 1correct
3gRNA Ga20ox 2correct
4Ga20ox consensus 1correct
5Ga20ox consensus 2correct
6Ga20ox knock-out 1-
7Ga20ox knock-out 2correct
8TFL consensus 1-
9TFL consensus 2-
10TFL knock-out 1correct
11TFL knock-out 2correct
12TFL gRNA 1correct
13TFL gRNA 2correct
141 Kb molecular weight markercorrect
  • 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.
LanesSamplesVerification
11 Kb molecular weight marker
2gRNA Ga20ox 1correct
3gRNA Ga20ox 2correct
4gRNA Ga20ox 3correct
5gRNA Ga20ox 4correct
6TFL knock-out 1correct
7TFL knock-out 2correct
81 Kb molecular weight markercorrect

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)
ReagentVolume(μL)ReagentVolume(μL)
TFL/Ga20ox gRNA1promoter 35s:5’region1
U6-261TFL/Ga20ox consensus TFL/Ga20ox knock-out1
psgRNA1luciferase1
3α11Tnos1
BSA10X1.23α11
Ligase Buffer1.2BSA10X1.2
BsaI1Ligase Buffer1.2
T4 ligase1BsmbI1
H2O milli-Q3.6T4 ligase1
H2O milli-Q2.6

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”.
LanesSamplesVerification
11 Kb molecular weight markercorrect
2gRNA TFL 1correct
3gRNA TFL 2correct
4TFL consensus 1correct
5TFL consensus 2correct
6TFL knock-out 1correct
7TFL knock-out 2correct
81 Kb molecular weight markercorrect
9gRNA Ga20ox 1correct
10gRNA Ga20ox 2correct
11Ga20ox consensus 1correct
12Ga20ox consensus 2-
13Ga20ox knock-out 1correct
14Ga20ox knock-out 2correct
151 Kb molecular weight markercorrect
  • 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:
ReagentVolume (μL)
Primer in order to sequence3
Miniprep reaction5
H2O milli-Q6
SequenceOrder
TFL gRNA210.13.201
Ga20 gRNA210.13.202
  • 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.
LanesSamplesVerification
11 Kb molecular weight markercorrect
2Target Ga20ox consensuscorrect
3Target Ga20ox Knock-outcorrect
4Target TFL consensuscorrect
5Target TFL knock-outcorrect
61 Kb molecular weight markercorrect

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:
LanesSamplesVerification
11 Kb molecular weight marker
2promoter 35s:5’ region:Target Ga20ox Rice:Luc:Tnos
3promoter 35s:5’ region:Target TFL Clemunules:Luc:Tnos
61 Kb molecular weight marker