Line 145: | Line 145: | ||
<tr><td>dNTPs</td><td>2</td><td>98°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°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°C | + | <tr><td>IG16JUN01</td><td>2.5</td><td>70°C</td><td>30 seconds</td></tr> |
− | <tr><td>IG16JUN02</td><td>2.5</td><td>72°C | + | <tr><td>IG16JUN02</td><td>2.5</td><td>72°C</td><td>1 minute 30 seconds</td></tr> |
<tr><td>Taq phusion</td><td>0.5</td><td>72°C</td><td>10 minutes</td></tr> | <tr><td>Taq phusion</td><td>0.5</td><td>72°C</td><td>10 minutes</td></tr> |
Revision as of 10:14, 26 July 2016
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:
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°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
- 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 |
01/07/2016
- 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.
Sample | DNA concentration (ng / μL) |
Clemenules1 | 3153.8 |
Clemenules 2 | 4527.9 |
- Perform a PCR to bind linker with luciferase:
Reagent | Volume(μL) | Program | ||
LuciferasepUPD | 1 | Temperature | Time | |
Buffer HF | 10 | 98°C | 5 minutes | |
dNTPs | 2 | 98°C | 35x | 30 seconds |
IG16JUN01 | 2.5 | 70°C | 30 seconds | |
IG16JUN02 | 2.5 | 72°C | 1 minute 30 seconds | |
Taq phusion | 0.5 | 72°C | 10 minutes | |
H2O milli-Q | 31.5 | 16°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.
- 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.
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.
- 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 from Goldenbraid Collection:
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 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.
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 | 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(μL) | 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 , 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)
Sample | DNA Concentration(ng / μL) | 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 , 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
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 plasmid | 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 plasmid | 1 | |
BSA10X | 1.2 | |
Ligase Buffer | 1.2 | |
BsaI | 1 | |
T4 ligase | 1 | |
H2O milli-Q | 3.6 |
- 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)
Reagent | Volume (μL) |
Promotor 35s:Cas9 : Tnos | 1 |
U6-26:TFL Clemenules sgRNA:psgRNA / U6-26:Ga20ox rice 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 37°C. 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 | correct |
2 | gRNA Ga20ox 1 | correct |
3 | gRNA Ga20ox 2 | correct |
4 | Ga20ox consensus 1 | correct |
5 | Ga20ox consensus 2 | correct |
6 | Ga20ox knock-out 1 | - |
7 | Ga20ox knock-out 2 | correct |
8 | TFL consensus 1 | - |
9 | TFL consensus 2 | - |
10 | TFL knock-out 1 | correct |
11 | TFL knock-out 2 | correct |
12 | TFL gRNA 1 | correct |
13 | TFL gRNA 2 | correct |
14 | 1 Kb molecular weight marker | correct |
- 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.
Lanes | Samples | Verification |
1 | 1 Kb molecular weight marker | |
2 | gRNA Ga20ox 1 | correct |
3 | gRNA Ga20ox 2 | correct |
4 | gRNA Ga20ox 3 | correct |
5 | gRNA Ga20ox 4 | correct |
6 | TFL knock-out 1 | correct |
7 | TFL knock-out 2 | correct |
8 | 1 Kb molecular weight marker | correct |
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)
Reagent | Volume(μL) | Reagent | Volume(μL) |
TFL/Ga20ox gRNA | 1 | promoter 35s: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 | 3α1 | 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
- 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”.
Lanes | Samples | Verification |
1 | 1 Kb molecular weight marker | correct |
2 | gRNA TFL 1 | correct |
3 | gRNA TFL 2 | correct |
4 | TFL consensus 1 | correct |
5 | TFL consensus 2 | correct |
6 | TFL knock-out 1 | correct |
7 | TFL knock-out 2 | correct |
8 | 1 Kb molecular weight marker | correct |
9 | gRNA Ga20ox 1 | correct |
10 | gRNA Ga20ox 2 | correct |
11 | Ga20ox consensus 1 | correct |
12 | Ga20ox consensus 2 | - |
13 | Ga20ox knock-out 1 | correct |
14 | Ga20ox knock-out 2 | correct |
15 | 1 Kb molecular weight marker | correct |
- 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:
Reagent | Volume (μL) |
Primer in order to sequence | 3 |
Miniprep reaction | 5 |
H2O milli-Q | 6 |
Sequence | Order |
TFL gRNA | 210.13.201 |
Ga20 gRNA | 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: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
- 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.
Lanes | Samples | Verification |
1 | 1 Kb molecular weight marker | correct |
2 | Target Ga20ox consensus | correct |
3 | Target Ga20ox Knock-out | correct |
4 | Target TFL consensus | correct |
5 | Target TFL knock-out | correct |
6 | 1 Kb molecular weight marker | correct |
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:
Lanes | Samples | Verification |
1 | 1 Kb molecular weight marker | |
2 | promoter 35s:5’ region:Target Ga20ox Rice:Luc:Tnos | |
3 | promoter 35s:5’ region:Target TFL Clemunules:Luc:Tnos | |
6 | 1 Kb molecular weight marker |