Team:Valencia UPV/Notebook
- May 18, 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.
- May 19, 2016
Refresh previously made culture by inoculating 10 μL in a new culture
medium.
- June 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:
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.
- June 15, 2016
Experiment is over due to snails haven’t eaten leafs enough so we have
not been able to see fluorescence.
- June 30, 2016
Orange DNA Genome Extraction protocol href
- Take from the glycerinates of GoldenBraid Collection:
| Plasmid | GB Code |
|---|---|
| omoter 35s:Cas9:nopaline synthase terminator (Tnos) | GB0639 |
| Luciferase (Luc) in pUPD2 | GB0096 |
| Tnos in pUPD2 | GB0037 |
- July 01, 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.
