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

 

18/05

Take glycerinated cultures of C58 Agrobacterium with dsRED from GoldenBraid Collection. Prepare broth culture (5 mL LB + 5 uL kanamycin + 5 uL Rifampin) 1:1000 and incubate overnight at 28^C.  

19/05

Refresh previously made culture by inoculating 10 uL in a new culture medium.

20/05

|Agroinfiltration path:#; in Nicotiana benthamiana of C58 with dsRED.

06/06

<l>Take from the glycerinates of Goldenbraid Collection:</l>

<t>  

  Plasmid GB Code

  pD6B3 alpha1 GB0015

  pD6B3 alpha2 GB0017

  pD6B3 omega1 GB0019

  pD6B3 omega2 GB0021

  pUPD2 GB0307

</t>

Prepare liquid culture (3 mL LB + 3 uL antibiotic) 1:1000. Incubate at 37^C overnight.  

<l>Experiment with snails:</l>

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

Experiment is over due to snails haven't eaten leafs enough so we have not been able to see fluorescence.

30/06

Orange DNA Genome Extraction protocol href

<l>Take from the glycerinates of GoldenBraid Collection:</l>

<t>

  Plasmid GB Code

  Promoter 35s:Cas9:nopaline synthase terminator (Tnos) GB0639

  Luciferase (Luc) in pUPD2 GB0096

  Tnos in pUPD2 GB0037

</t>

01/07

<l>

Miniprep with E.Z.N.A. Plasmid Mini Kit I, Q(capless) Spin of:  

<l>Promoter 35s:Cas9 : Tnos</l>

<l>Luciferase and nopaline synthase terminator cultures haven't succeed. Repeat Luc and Tnos cultures. </l>

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

<l>

Miniprep with E.Z.N.A. Plasmid Mini Kit I, Q(capless) Spin of:

<l>

Luc in pUPD2

<l>Tnos in pUPD2</l>

<l>Check orange DNA genome concentration with NanoDrop. </l>

<t>

  Sample DNA concentration (ng / uL)

  Clemenules1 3153.8

  Clemenules 2 4527.9

</t>

<l>Perform a PCR to bind linker with luciferase:</l>

<t>

  Reagent Volume(uL) 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 &infin;

</t>

04/07

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 href

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 --> Linker:Luciferase into a pUPD2

Transform E.coli DH5alpha with it. The method that is necessary to carry out this procedure is explained in |protocols path:#;

Check DNA concentration with NanoDrop.  

<t>

  SAMPLE DNA Concentration(ng / uL)

  Rice Gleva 1 22.8

  Rice Gleva 2 17.3

</t>

05/07

Run electrophoresis gel of Gleva rice DNA. We have checked that there isn't DNA.  

Repeat: Rice DNA Genome Extraction href

Check DNA concentration with NanoDrop.

<t>

  SAMPLE DNA Concentration(ng / uL)

  Rice Gleva 1 294.9

  Rice Gleva 2 193.7

</t>

Run electrophoresis gel of Gleva rice DNA. It observed that genome extraction is correctly done.

06/07  

Take glycerinated cultures from Goldenbraid Collection:

<t>

  GB part Plasmid Antibiotic Number GB

  psgRNA pUPD Ampicillin 0645

  U6-26 pUPD Ampicillin 1001

</t>

07/07

<l>

Miniprep with E.Z.N.A. Plasmid Mini Kit I, Q(capless) Spin of:

<l>

psgRNA in pUPD2

<l>U6-26 in pUPD2</l>

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 path:#;

<t>

  Sample Initial concentration(ng/uL) Final concentration(ng/uL) Initial volume(uL) Final volume (uL)

  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

</t>

<t>

  REAGENT VOLUME(uL) 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 &infin;

</t>

<t>

  REAGENT VOLUME(uL) 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 &infin;

</t>

Ligate reaction of promoter 35s:5' region in pUPD2. Following ligation protocol href, BsmbI enzyme is used in this reaction.

08/07

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 (alpha1 and kanamycin)

09/07

<l>

Miniprep with E.Z.N.A. Plasmid Mini Kit I, Q(capless) Spin of:

<l>promoter 35s:GFP:Tnos</l>

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

Pick a single E. coli DH5alpha (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 uL 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)

<t>

  Sample DNA Concentration(ng / uL) DNA Concentration(ng)

  1 105.2 5035.2

  2 104.6 5035.2

</t>

Targets ligations in pUPD2 (Orange Clemenules and Rice Gleva). Following |ligation protocol path:#;, BsmbI enzyme is used in this reaction.

12/07

Pick a single E. coli DH5alpha (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 uL of chloramphenicol in a 50 ml tube with the colony and incubate it 16 hours at 37^C.

<l>

Miniprep with E.Z.N.A. Plasmid Mini Kit I, Q(capless) Spin of:  

<l>Promoter 35s:5'region in pUPD2</l>

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.

<l>

Miniprep with E.Z.N.A. Plasmid Mini Kit I, Q(capless) Spin of:  

<l>

Rice Gleva target in pUPD2

<l>Orange Clemenules target in pUPD2</l>

Digestion of minipreps with NotI. Incubate 1 hour at 37^C.

<l>

Run electrophoresis gel of the same devise:

<l>

Rice Gleva target in pUPD2

<l>Orange Clemenules target in pUPD2</l>

<l>

Ligation using Golden Braid assembly of the next devise:

<l>Promoter 35s:5' region:Target:Luc:Tnos in alpha1 plasmid.</l>