|
|
| Line 9: |
Line 9: |
| | <p> | | <p> |
| | | | |
| − |
| |
| − | 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 ºC.
| |
| − | 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. (ENLACE)
| |
| − | 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 ºC 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.
| |
| − | 15/06/2016
| |
| − | Experiment is over due to snails haven’t eaten leafs enough so we haven’t been able to see fluorescence.
| |
| − | 30/06/2016
| |
| − | Orange DNA Genome Extraction protocol (ENLACE)
| |
| − | Take from the glycerinates of GoldenBraid Collection:
| |
| − | Plasmid
| |
| − | GB Code
| |
| − | Promoter 35S : Cas9 : nopaline synthase terminator (T-nos)
| |
| − | GB0639
| |
| − | Luciferase (Luc)
| |
| − | GB0096
| |
| − | T-nos
| |
| − | GB0037
| |
| − |
| |
| − |
| |
| − | 01/07/2016
| |
| − | Miniprep with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of:
| |
| − | Promoter 35S : Cas9 : T-nos
| |
| − |
| |
| − | 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 and Tnos.
| |
| − |
| |
| − | Check orange DNA genome concentration with NanoDrop.
| |
| − | Sample
| |
| − | DNA concentration (ng / μL)
| |
| − | Clemenules 1
| |
| − | 3153.8
| |
| − | Clemenules 2
| |
| − | 4527.9
| |
| − |
| |
| − | Perform a PCR to bind linker with luciferase
| |
| − | Reagent
| |
| − | Volume (μL)
| |
| − | Program
| |
| − | Luciferase pUPD
| |
| − | 1
| |
| − | Temperature
| |
| − | Time
| |
| − | Buffer HF
| |
| − | 10
| |
| − | 98 ºC
| |
| − | 5 minutes
| |
| − | dNTPs
| |
| − | 2
| |
| − | 98 ºC
| |
| − | 35x
| |
| − | 30 seconds
| |
| − | IG16JUN01
| |
| − | 2.5
| |
| − | 70 ºC
| |
| − | 30 seconds
| |
| − | IG16JUN02
| |
| − | 2.5
| |
| − | 72 ºC
| |
| − | 1 minute 30 seconds
| |
| − | Taq phusion
| |
| − | 0.5
| |
| − | 72 ºC
| |
| − | 10 minutes
| |
| − | H2O milli-Q
| |
| − | 31.5
| |
| − | 16 ºC
| |
| − | ∞
| |
| − |
| |
| − | 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 (ENLACE)
| |
| − |
| |
| − | 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 linker: Luciferase into a pUPD2 and 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:
| |
| − | 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 and U6-26
| |
| − |
| |
| − | 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.
| |
| − |
| |
| − | 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
| |
| − | 98ºC
| |
| − | 5 minutes
| |
| − | dNTPs
| |
| − | 2
| |
| − | 98ºC
| |
| − | 35x
| |
| − | 30 seconds
| |
| − | IG16JUL01 (TFL_For)
| |
| − | 2.5
| |
| − | 64ºC
| |
| − | 30 seconds
| |
| − | IG16JUL02 (TFL_Rev)
| |
| − | 2.5
| |
| − | 72ºC
| |
| − | 30 seconds
| |
| − | Taq phusion
| |
| − | 0.5
| |
| − | 72ºC
| |
| − | 10 minutes
| |
| − | H2O milli-Q
| |
| − | 31.5
| |
| − | 16ºC
| |
| − | ∞
| |
| − |
| |
| − | REAGENT
| |
| − | VOLUME (μL)
| |
| − | PROGRAM
| |
| − | Gleva DNA
| |
| − | 1
| |
| − | TEMPERATURE
| |
| − | TIME
| |
| − | Buffer HF
| |
| − | 10
| |
| − | 98ºC
| |
| − | 5 minutes
| |
| − | dNTPs
| |
| − | 2
| |
| − | 98ºC
| |
| − | 35x
| |
| − | 30 seconds
| |
| − | IG16JUL03 (Ga20_for)
| |
| − | 2.5
| |
| − | 72ºC
| |
| − | 30 seconds
| |
| − | IG16JUL02 (Ga20_rev)
| |
| − | 2.5
| |
| − | 72ºC
| |
| − | 30 seconds
| |
| − | Taq phusion
| |
| − | 0.5
| |
| − | 72ºC
| |
| − | 10 minutes
| |
| − | H2O milli-Q
| |
| − | 31.5
| |
| − | 16ºC
| |
| − | ∞
| |
| − |
| |
| − |
| |
| − | Ligate 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 construction: promoter 35S : 5’ region (electroporation 2.5KV). Plating it.
| |
| − |
| |
| − | Take glycerinated culture for Georgia collaboration. The construction 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 35S : 5’ region petri dishes. Repeat transformation procedure and plating again.
| |
| − | 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 37ºC 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
| |
| − |
| |
| − | Targets ligations ( 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 37ºC.
| |
| − |
| |
| − | 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 37ºC
| |
| − |
| |
| − | Run electrophoresis gel of the construction: 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 37ºC.
| |
| − |
| |
| − | Run electrophoresis gel of the same construction:
| |
| − | Rice Gleva target in pUPD2
| |
| − | Orange Clemenules target in pUPD2
| |
| − |
| |
| − | Ligation using Golden Braid assembly of the next construction:
| |
| − | Promoter 35S : 5’ region : Target : LUC : Tnos in α1
| |
| − | 13/07/2016
| |
| − | E.coli Transformation with the construction:
| |
| − | Promoter 35S : 5’ region : Target : LUC : Tnos in α1
| |
| − |
| |
| − | E. coli plating in plates with Kanamycin (antibiotic). Incubate overnight at 37ºC
| |
| − | 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 37ºC.
| |
| − |
| |
| − | Primers IG16JUL09, IG16JUL10, IG16JUL11, IG16JUL12, IG16JUL13, IG16JUL14, IG16JUL15 and IG16JUL16 arrived.
| |
| − |
| |
| − | Ligation using Golden Braid assembly of the next constructions:
| |
| − | Promoter 35S : 5’Region : Clemenules target control + : Luc : Tnos
| |
| − | Promoter 35S : 5’Region : Gleva target control + : 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 1μM. It is necessary taking to 10 μM.
| |
| − | 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
| |
| − | 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
| |
| − | 1
| |
| − | BSA10X
| |
| − | 1.2
| |
| − | Ligase Buffer
| |
| − | 1.2
| |
| − | BsaI
| |
| − | 1
| |
| − | T4 ligase
| |
| − | 1
| |
| − | H2O milli-Q
| |
| − | 3.6
| |
| − |
| |
| − | E.coli Transformation with the constructions previously explained.
| |
| − |
| |
| − | E. coli plating in 6 plates (6 ligation reactions) with Kanamycin (antibiotic). Incubate overnight at 37ºC.
| |
| − |
| |
| − | 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 37ºC
| |
| − | 15/07/2016
| |
| − | Ligation using Golden Braid assembly of the next construction:
| |
| − | Promotor 35S : Cas9 : Tnos – Promotor 35S : 5’ region : CL target : Luc : Tnos
| |
| − | Promotor 35S : Cas9 : Tnos – promotor 35S : 5’ region : A target : Luc : Tnos
| |
| − |
| |
| − | E. coli Transformation with the constructions previously explained.
| |
| − |
| |
| − | Run electrophoresis gel of the products from the digestion of minipreps. The constructions are:
| |
| − | Promoter 35S : 5’ region : CL target : Luc : Tnos (3α1)
| |
| − | Promoter 35S : 5’ region : A target : Luc : Tnos (3α1)
| |
| − |
| |
| − | Sequencing products:
| |
| − | Promotor 35S : 5’ region in pUPD2 → correct
| |
| − | 16/07/2016
| |
| − |
| |
| − | E.coli transformations with:
| |
| − | Promoter 35S : 5’ region : CL target : Luc : Tnos (3α1)
| |
| − | Promoter 35S : 5’ region : A target : Luc : Tnos (3α1)
| |
| − |
| |
| − | Plating the last constructions in 2 plates with Agrobacterium C58. Incubate 2 days at 28ºC.
| |
| − |
| |
| − | Minipreps (2 samples for each construction) with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of:
| |
| − | Promoter 35S : 5’Region : Clemenules target control + : Luc : Tnos
| |
| − | Promoter 35S : 5’Region : Gleva target control + : 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 construction:
| |
| − | Promotor 35S : Cas9 : Tnos –U6 : CL sgRNA : psgRNA (scaffold) (Ω1)
| |
| − | Promotor 35S : Cas9 : Tnos –U6 : A sgRNA : psgRNA (scaffold) (Ω1)
| |
| − | Reagent
| |
| − | Volume (μL)
| |
| − | Promotor 35S : Cas9 : Tnos
| |
| − | 1
| |
| − | U6-26 : CL sgRNA : psgRNA / U6-26 : A 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 37ºC. 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
| |
| − |
| |
| − | 2
| |
| − | gRNA Ga20ox 1
| |
| − |
| |
| − | 3
| |
| − | gRNA Ga20ox 2
| |
| − |
| |
| − | 4
| |
| − | Ga20ox consensus 1
| |
| − |
| |
| − | 5
| |
| − | Ga20ox consensus 2
| |
| − |
| |
| − | 6
| |
| − | Ga20ox knock-out 1
| |
| − |
| |
| − |
| |
| − | 7
| |
| − | Ga20ox knock-out 2
| |
| − |
| |
| − | 8
| |
| − | TFL consensus 1
| |
| − |
| |
| − |
| |
| − | 9
| |
| − | TFL consensus 2
| |
| − |
| |
| − |
| |
| − | 10
| |
| − | TFL knock-out 1
| |
| − |
| |
| − | 11
| |
| − | TFL knock-out 2
| |
| − |
| |
| − | 12
| |
| − | TFL gRNA 1
| |
| − |
| |
| − | 13
| |
| − | TFL gRNA 2
| |
| − |
| |
| − | 14
| |
| − | 1 Kb molecular weight marker
| |
| − |
| |
| − |
| |
| − | Pick a single E. coli DH5α colony from the plates that have been incubated overnight. The constructions are:
| |
| − | Promotor 35S : TFL Knock-out : Luc : Tnos (4 samples)
| |
| − | Promoter U6-26 : A sgRNA : psgRNA (2 samples)
| |
| − | 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 37ºC.
| |
| − |
| |
| − | 17/07/2016
| |
| − | DH5α E.coli transformations with:
| |
| − | Promotor 35S : Cas9 : Tnos –U6 : CL sgRNA : psgRNA (scaffold) (Ω1)
| |
| − | Promotor 35S : Cas9 : Tnos –U6 : A sgRNA : psgRNA (scaffold) (Ω1)
| |
| − |
| |
| − | Plating E.coli transformations in plates with LB + agar + IPTG + XGal and incubate it overnight at 37ºC.
| |
| − |
| |
| − | Miniprep with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of:
| |
| − | Promoter U6-26 : A 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 37ºC. There is a total of six 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
| |
| − |
| |
| − | 2
| |
| − | gRNA Ga20ox 1
| |
| − |
| |
| − | 3
| |
| − | gRNA Ga20ox 2
| |
| − |
| |
| − | 4
| |
| − | gRNA Ga20ox 3
| |
| − |
| |
| − | 5
| |
| − | gRNA Ga20ox 4
| |
| − |
| |
| − | 6
| |
| − | TFL knock-out 1
| |
| − |
| |
| − | 7
| |
| − | TFL knock-out 2
| |
| − |
| |
| − | 8
| |
| − | 1 Kb molecular weight marker
| |
| − |
| |
| − |
| |
| − | However, despite the fact that electrophoresis gel have correctly run, we have decided to repeat the ligation reactions. We haven’t get the expected results for a gRNA.
| |
| − |
| |
| − | Ligation using Golden Braid assembly of the next constructions:
| |
| − | Promoter 35S : 5’Region : Clemenules target control + : Luc : Tnos
| |
| − | Promoter 35S : 5’Region : Gleva target control + : 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)
| |
| − | Reagent
| |
| − | Volume (μL)
| |
| − | Reagent
| |
| − | Volume (μL)
| |
| − | TFL/Ga20ox gRNA
| |
| − | 1
| |
| − | Promotor35S :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
| |
| − | 3Ω1
| |
| − | 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
| |
| − | DH5α E.coli transformations 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 37ºC.
| |
| − |
| |
| − | Pick a single Agrobacterium C58 colony from the plates that have been incubating since 16/06/2016. The constructions are:
| |
| − | Promoter 35S : 5’ region : CL target : Luc : Tnos (3α1)
| |
| − | Promoter 35S : 5’ region : A target : Luc : Tnos (3α1)
| |
| − | Incubate it 48 hours at 28ºC.
| |
| − | 19/07/2016
| |
| − |
| |
| − | Pick transformed E.coli colony from the incubated plates. The constructions are:
| |
| − | Promoter 35S : 5’Region : Clemenules target control + : Luc : Tnos
| |
| − | Promoter 35S : 5’Region : Gleva target control + : 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)
| |
| − | Incubate it overnight at 37ºC.
| |
| − | 20/07/2016
| |
| − |
| |
| − | - Miniprep with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of:
| |
| − | § Promoter 35S : 5’Region : Clemenules target knockout : Luc : Tnos
| |
| − | § Promoter 35S : 5’Region : Gleva target control knockout : 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)
| |
| − |
| |
| − | - Digestion of the minipreps with EcoRI which have been done before. Incubate 1 hour at 37ºC. There is a total of six minipreps. It has been followed the Miniprep digestion protocol.
| |
| − |
| |
| − | - Run electrophoresis gel of the digestion products. We will keep the minipreps with “1”.
| |
| − | Lanes
| |
| − | Samples
| |
| − | Verification
| |
| − | 1
| |
| − | 1 Kb molecular weight marker
| |
| − |
| |
| − |
| |
| − | 2
| |
| − | gRNA TFL 1
| |
| − |
| |
| − |
| |
| − | 3
| |
| − | gRNA TFL 2
| |
| − |
| |
| − |
| |
| − | 4
| |
| − | TFL consensus 1
| |
| − |
| |
| − |
| |
| − | 5
| |
| − | TFL consensus 2
| |
| − |
| |
| − |
| |
| − | 6
| |
| − | TFL knock-out 1
| |
| − |
| |
| − |
| |
| − | 7
| |
| − | TFL knock-out 2
| |
| − |
| |
| − |
| |
| − | 8
| |
| − | 1 Kb molecular weight marker
| |
| − |
| |
| − |
| |
| − | 9
| |
| − | gRNA Ga20ox 1
| |
| − |
| |
| − |
| |
| − | 10
| |
| − | gRNA Ga20ox 2
| |
| − |
| |
| − |
| |
| − | 11
| |
| − | Ga20ox consensus 1
| |
| − |
| |
| − |
| |
| − | 12
| |
| − | Ga20ox consensus 2
| |
| − |
| |
| − | 13
| |
| − | Ga20ox knock-out 1
| |
| − |
| |
| − |
| |
| − | 14
| |
| − | Ga20ox knock-out 2
| |
| − |
| |
| − |
| |
| − | 15
| |
| − | 1 Kb molecular weight marker
| |
| − |
| |
| − |
| |
| − |
| |
| − | - Ligation using Golden Braid assembly of the next constructions. Is used the restriction enzyme BsmbI.
| |
| − | § Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA
| |
| − | § Promoter 35s:Cas 9:T-nos – U6-26:Ga20ox sgRNA: psgRNA
| |
| − | 21/07/2016
| |
| − |
| |
| − | - E.coli transformations with:
| |
| − | § Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA
| |
| − | § Promoter 35s:Cas 9:T-nos – U6-26:Ga20ox sgRNA: psgRNA
| |
| − | Incubate 2 hours at 37 ºC.
| |
| − | - Plating E.coli transformation explained before and incubate it at 37ºC overnight.
| |
| − |
| |
| − | - Agrobacterium C58 transformation with:
| |
| − | § Promoter 35S : 5’Region : Clemenules target knockout : Luc : Tnos
| |
| − | § Promoter 35S : 5’Region : Gleva target control knockout : Luc : Tnos
| |
| − | § Promoter 35S : 5’Region : Clemenules target consensus : Luc : Tnos
| |
| − | § Promoter 35S : 5’Region : Gleva target consensus : Luc : Tnos
| |
| − | Incubate 48 hours at 28 ºC.
| |
| − |
| |
| − | 22/07/2016
| |
| − |
| |
| − | - Sequencing reaction
| |
| − | Reagent
| |
| − | Volume (μL)
| |
| − | Primer to sequence
| |
| − | 3
| |
| − | Miniprep reaction
| |
| − | 5
| |
| − | H2O milli-Q
| |
| − | 6
| |
| − |
| |
| − | TFL gRNA à 210.13.201
| |
| − | Ga20 gRNA à 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:T-nos – U6-26:TFL sgRNA: psgRNA
| |
| − |
| |
| − | - Plating the last construction in plates with Agrobacterium C58. Plates contain spec + IPTG + X-Gal. Incubate 2 days at 28ºC.
| |
| − |
| |
| − | - Pick a single E.coli DH5α (Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA and Promoter 35s:Cas 9:T-nos – 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 37ºC with shaking.
| |
| − |
| |
| − | 23/07/2016
| |
| − |
| |
| − | - Minipreps (4 samples for each construction) with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of:
| |
| − | § Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA
| |
| − | § Promoter 35s:Cas 9:T-nos – U6-26:Ga20ox sgRNA: psgRNA
| |
| − |
| |
| − | - Digestion of minipreps with BamHI following digestion protocol. Incubate 1 hour at 37ºC.
| |
| − |
| |
| − | - Run 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:T-nos – U6-26:TFL sgRNA: psgRNA and Promoter 35s:Cas 9:T-nos – 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 28ºC with shaking.
| |
| − |
| |
| − | - Agrobacterium C58 transformations with:
| |
| − | § Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA
| |
| − | § Promoter 35s:Cas 9:T-nos – U6-26:Ga20ox sgRNA: psgRNA
| |
| − | 24/07/2016
| |
| − |
| |
| − | - Store the next cultures at -80 ºC:
| |
| − | § Promoter 35s:5’ region in pUPD2 (DH5α) à 1
| |
| − | § Linker: luciferase in pUPD2 (DH5α) à 2
| |
| − | 25/07/2016
| |
| − |
| |
| − | - Pick a single Agrobacterium C58 (Promoter 35s:Cas 9:T-nos – U6-26:TFL sgRNA: psgRNA in 3Ω1 and promoter 35s:Cas 9:T-nos – 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 28ºC with shaking.
| |
| − |
| |
| − | Incubate it 48 hours at 28 ºC
| |
| − |
| |
| − | - Minipreps of Agrobacterium with E.Z.N.A.® Plasmid Mini Kit I, Q(capless) Spin of:
| |
| − | § Promoter 35S : 5’Region : Clemenules target control + : Luc : Tnos
| |
| − | § Promoter 35S : 5’Region : Gleva target control + : Luc : Tnos
| |
| − | § Promoter 35S : 5’Region : Clemenules target consensus : Luc : Tnos
| |
| − | § Promoter 35S : 5’Region : Gleva target consensus : Luc : Tnos
| |
| − |
| |
| − | - Digestion of minipreps with the restriction enzyme EcoRI. Incubate 1 hour at 37ºC.
| |
| − |
| |
| − | - Run electrophoresis gel of the digestion products.
| |
| − |
| |
| − | Lanes
| |
| − | Samples
| |
| − | Verification
| |
| − | 1
| |
| − | 1 Kb molecular weight marker
| |
| − |
| |
| − |
| |
| − | 2
| |
| − | Target Ga20ox consensus
| |
| − |
| |
| − |
| |
| − | 3
| |
| − | Target Ga20ox Knockout
| |
| − |
| |
| − |
| |
| − | 4
| |
| − | Target TFL consensus
| |
| − |
| |
| − |
| |
| − | 5
| |
| − | Target TFL knockout
| |
| − |
| |
| − |
| |
| − | 6
| |
| − | 1 Kb molecular weight marker
| |
| | | | |
| | </p> | | </p> |
