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
Take from the glycerinates of Goldenbraid Collection:
Plasmid GB Code
pD6B3 alpha1 GB0015
pD6B3 alpha2 GB0017
pD6B3 omega1 GB0019
pD6B3 omega2 GB0021
pUPD2 GB0307
Prepare liquid culture (3 mL LB + 3 uL antibiotic) 1:1000. Incubate at 37^C 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
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
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
Miniprep with E.Z.N.A. Plasmid Mini Kit I, Q(capless) Spin of:
Promoter 35s:Cas9 : TnosLuciferase 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
Miniprep with E.Z.N.A. Plasmid Mini Kit I, Q(capless) Spin of:
Luc in pUPD2
Tnos in pUPD2Check orange DNA genome concentration with NanoDrop.
Sample DNA concentration (ng / uL)
Clemenules1 3153.8
Clemenules 2 4527.9
Perform a PCR to bind linker with luciferase:
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 ∞
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.
SAMPLE DNA Concentration(ng / uL)
Rice Gleva 1 22.8
Rice Gleva 2 17.3
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.
SAMPLE DNA Concentration(ng / uL)
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
Take glycerinated cultures from Goldenbraid Collection:
GB part Plasmid Antibiotic Number GB
psgRNA pUPD Ampicillin 0645
U6-26 pUPD Ampicillin 1001
07/07
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 path:#;
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
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 ∞
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 ∞
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
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
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)
Sample DNA Concentration(ng / uL) 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 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.
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 alpha1 plasmid.
