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

Line 501: Line 501:
 
<tr><td></td><td>Reagent</td><td>Volume (&mu;L)</td></tr>
 
<tr><td></td><td>Reagent</td><td>Volume (&mu;L)</td></tr>
  
−
<tr><td></td><td>Clemenules target control + / Gleva target control + / Clemenules target consensus / Gleva target consensus</td><td>1 </td></tr>
+
<tr><td></td><td>Target control positive / Target consensus</td><td>1 </td></tr>
  
−
<tr><td></td><td>Promoter 35s:5&rsquo; region</td><td>1</td></tr>
+
<tr><td></td><td>promoter 35s:5&rsquo; region</td><td>1</td></tr>
  
 
<tr><td></td><td>Luciferase</td><td>1</td></tr>
 
<tr><td></td><td>Luciferase</td><td>1</td></tr>
Line 533: Line 533:
 
<tr><td></td><td>sgRNA Clemenules / sgRNA Gleva</td><td>1</td></tr>
 
<tr><td></td><td>sgRNA Clemenules / sgRNA Gleva</td><td>1</td></tr>
  
−
<tr><td></td><td>Promoter U6 -26</td><td>1</td></tr>
+
<tr><td></td><td>promoter U6 -26</td><td>1</td></tr>
  
 
<tr><td></td><td>psgRNA (scaffold)</td><td>1</td></tr>
 
<tr><td></td><td>psgRNA (scaffold)</td><td>1</td></tr>
Line 679: Line 679:
 
<tr><td>1</td><td>1 Kb molecular weight marker</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
 
<tr><td>1</td><td>1 Kb molecular weight marker</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>2</td><td>gRNA Ga20ox 1</td><td>correct</td></tr>
+
<tr><td>2</td><td>gRNA Ga20ox 1</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>3</td><td>gRNA Ga20ox 2</td><td>correct</td></tr>
+
<tr><td>3</td><td>gRNA Ga20ox 2</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>4</td><td>Ga20ox consensus 1</td><td>correct</td></tr>
+
<tr><td>4</td><td>Ga20ox consensus 1</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png">v</td></tr>
  
−
<tr><td>5</td><td>Ga20ox consensus 2</td><td>correct</td></tr>
+
<tr><td>5</td><td>Ga20ox consensus 2</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
 
<tr><td>6</td><td>Ga20ox knock-out 1</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/7/7a/T--Valencia_UPV--cross.png"></td></tr>
 
<tr><td>6</td><td>Ga20ox knock-out 1</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/7/7a/T--Valencia_UPV--cross.png"></td></tr>
  
−
<tr><td>7</td><td>Ga20ox knock-out 2</td><td>correct</td></tr>
+
<tr><td>7</td><td>Ga20ox knock-out 2</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>8</td><td>TFL consensus 1</td><td>-</td></tr>
+
<tr><td>8</td><td>TFL consensus 1</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/7/7a/T--Valencia_UPV--cross.png"></td></tr>
  
−
<tr><td>9</td><td>TFL consensus 2</td><td>-</td></tr>
+
<tr><td>9</td><td>TFL consensus 2</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/7/7a/T--Valencia_UPV--cross.png"></td></tr>
  
−
<tr><td>10</td><td>TFL knock-out 1</td><td>correct</td></tr>
+
<tr><td>10</td><td>TFL knock-out 1</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>11</td><td>TFL knock-out 2</td><td>correct</td></tr>
+
<tr><td>11</td><td>TFL knock-out 2</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>12</td><td>TFL gRNA 1</td><td>correct</td></tr>
+
<tr><td>12</td><td>TFL gRNA 1</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>13</td><td>TFL gRNA 2</td><td>correct</td></tr>
+
<tr><td>13</td><td>TFL gRNA 2</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>14</td><td>1 Kb molecular weight marker</td><td>correct</td></tr>
+
<tr><td>14</td><td>1 Kb molecular weight marker</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
 
</div></table>
 
</div></table>
Line 757: Line 757:
 
<tr><td>Lanes</td><td>Samples</td><td>Verification</td></tr>
 
<tr><td>Lanes</td><td>Samples</td><td>Verification</td></tr>
  
−
<tr><td>1</td><td>1 Kb molecular weight marker</td></tr>
+
<tr><td>1</td><td>1 Kb molecular weight marker</td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></tr>
  
−
<tr><td>2</td><td>gRNA Ga20ox 1</td><td>correct</td></tr>
+
<tr><td>2</td><td>gRNA Ga20ox 1</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>3</td><td>gRNA Ga20ox 2</td><td>correct</td></tr>
+
<tr><td>3</td><td>gRNA Ga20ox 2</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>4</td><td>gRNA Ga20ox 3</td><td>correct</td></tr>
+
<tr><td>4</td><td>gRNA Ga20ox 3</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>5</td><td>gRNA Ga20ox 4</td><td>correct</td></tr>
+
<tr><td>5</td><td>gRNA Ga20ox 4</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>6</td><td>TFL knock-out 1</td><td>correct</td></tr>
+
<tr><td>6</td><td>TFL knock-out 1</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>7</td><td>TFL knock-out 2</td><td>correct</td></tr>
+
<tr><td>7</td><td>TFL knock-out 2</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>8</td><td>1 Kb molecular weight marker</td><td>correct</td></tr>
+
<tr><td>8</td><td>1 Kb molecular weight marker</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
 
</div></table>
 
</div></table>
Line 909: Line 909:
 
<tr><td>Lanes</td><td>Samples</td><td>Verification</td></tr>
 
<tr><td>Lanes</td><td>Samples</td><td>Verification</td></tr>
  
−
<tr><td>1</td><td>1 Kb molecular weight marker</td><td>correct</td></tr>
+
<tr><td>1</td><td>1 Kb molecular weight marker</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>2</td><td>gRNA TFL 1</td><td>correct</td></tr>
+
<tr><td>2</td><td>gRNA TFL 1</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>3</td><td>gRNA TFL 2</td><td>correct</td></tr>
+
<tr><td>3</td><td>gRNA TFL 2</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>4</td><td>TFL consensus 1</td><td>correct</td></tr>
+
<tr><td>4</td><td>TFL consensus 1</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>5</td><td>TFL consensus 2</td><td>correct</td></tr>
+
<tr><td>5</td><td>TFL consensus 2</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>6</td><td>TFL knock-out 1</td><td>correct</td></tr>
+
<tr><td>6</td><td>TFL knock-out 1</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>7</td><td>TFL knock-out 2</td><td>correct</td></tr>
+
<tr><td>7</td><td>TFL knock-out 2</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>8</td><td>1 Kb molecular weight marker</td><td>correct</td></tr>
+
<tr><td>8</td><td>1 Kb molecular weight marker</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>9</td><td>gRNA Ga20ox 1</td><td>correct</td></tr>
+
<tr><td>9</td><td>gRNA Ga20ox 1</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>10</td><td>gRNA Ga20ox 2</td><td>correct</td></tr>
+
<tr><td>10</td><td>gRNA Ga20ox 2</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>11</td><td>Ga20ox consensus 1</td><td>correct</td></tr>
+
<tr><td>11</td><td>Ga20ox consensus 1</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>12</td><td>Ga20ox consensus 2</td><td>-</td></tr>
+
<tr><td>12</td><td>Ga20ox consensus 2</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/7/7a/T--Valencia_UPV--cross.png"></td></tr>
  
−
<tr><td>13</td><td>Ga20ox knock-out 1</td><td>correct</td></tr>
+
<tr><td>13</td><td>Ga20ox knock-out 1</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>14</td><td>Ga20ox knock-out 2</td><td>correct</td></tr>
+
<tr><td>14</td><td>Ga20ox knock-out 2</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>15</td><td>1 Kb molecular weight marker</td><td>correct</td></tr>
+
<tr><td>15</td><td>1 Kb molecular weight marker</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
 
</div></table>
 
</div></table>
Line 1,125: Line 1,125:
 
<tr><td>Lanes</td><td>Samples</td><td>Verification</td></tr>
 
<tr><td>Lanes</td><td>Samples</td><td>Verification</td></tr>
  
−
<tr><td>1</td><td>1 Kb molecular weight marker</td><td>correct</td></tr>
+
<tr><td>1</td><td>1 Kb molecular weight marker</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>2</td><td>Target Ga20ox consensus</td><td>correct</td></tr>
+
<tr><td>2</td><td>Target Ga20ox consensus</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>3</td><td>Target Ga20ox Knock-out</td><td>correct</td></tr>
+
<tr><td>3</td><td>Target Ga20ox Knock-out</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>4</td><td>Target TFL consensus</td><td>correct</td></tr>
+
<tr><td>4</td><td>Target TFL consensus</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>5</td><td>Target TFL knock-out</td><td>correct</td></tr>
+
<tr><td>5</td><td>Target TFL knock-out</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
−
<tr><td>6</td><td>1 Kb molecular weight marker</td><td>correct</td></tr>
+
<tr><td>6</td><td>1 Kb molecular weight marker</td><td><img class="check_img" src="https://static.igem.org/mediawiki/2016/b/b7/T--Valencia_UPV--check.png"></td></tr>
  
 
</div></table>
 
</div></table>

Revision as of 10:01, 27 July 2016

Home


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)
Target control positive / 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 marker
2gRNA Ga20ox 1
3gRNA Ga20ox 2
4Ga20ox consensus 1v
5Ga20ox consensus 2
6Ga20ox knock-out 1
7Ga20ox knock-out 2
8TFL consensus 1
9TFL consensus 2
10TFL knock-out 1
11TFL knock-out 2
12TFL gRNA 1
13TFL gRNA 2
141 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.

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 1
3gRNA Ga20ox 2
4gRNA Ga20ox 3
5gRNA Ga20ox 4
6TFL knock-out 1
7TFL knock-out 2
81 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
    • 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 marker
2gRNA TFL 1
3gRNA TFL 2
4TFL consensus 1
5TFL consensus 2
6TFL knock-out 1
7TFL knock-out 2
81 Kb molecular weight marker
9gRNA Ga20ox 1
10gRNA Ga20ox 2
11Ga20ox consensus 1
12Ga20ox consensus 2
13Ga20ox knock-out 1
14Ga20ox knock-out 2
151 Kb molecular weight marker
  • 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 marker
2Target Ga20ox consensus
3Target Ga20ox Knock-out
4Target TFL consensus
5Target TFL knock-out
61 Kb molecular weight marker

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