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Team:Osaka/week5
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(New page: ==August 22 (Sun)== # Transfer of 001 to culture solution; incubation at 30°C (''why??'') # Transformation of the following parts (See Table 4) #* O/N incubation at 37°C as per normal {|...) |
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# Transfer of 001 to culture solution; incubation at 30°C (''why??'') | # Transfer of 001 to culture solution; incubation at 30°C (''why??'') | ||
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#* 1-1K, 1-1I, 3-3G (yesterday's transformations) | #* 1-1K, 1-1I, 3-3G (yesterday's transformations) | ||
#* 2-4A, 3-11I (repeat pick-up from 10/22, 10/23 plates) | #* 2-4A, 3-11I (repeat pick-up from 10/22, 10/23 plates) | ||
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| + | [[Team:Osaka/Notebook|Back to Notebook]] | ||
Latest revision as of 09:16, 13 October 2010
August 22 (Sun)
- Transfer of 001 to culture solution; incubation at 30°C (why??)
- Transformation of the following parts (See Table 4)
- O/N incubation at 37°C as per normal
| ID | Part Name | Resistance | Description |
|---|---|---|---|
| 2-4A | <bbpart>BBa_J63005</bbpart> | A | yeast ADH1 promoter |
| F1 | N/A | A | beta-galactosidase from Edinburgh team |
| F2 | N/A | C | RBS + F1 |
| F3 | N/A | C | Lac promoter + RBS + F1 |
August 23 (Mon)
- Transfer of 2-4A, F1, F2, F3 transformed yesterday to solution culture
- Miniprep & 'Cut check' of 001
- cut at EcoRI, SpeI
- gel run with DNA ladder, digested plasmid, undigested plasmid
- (RESULTS?)
- Transfer of 3 more colonies of 001 to liquid solution (to store as glycerol stock - see Tue notes)
- Transformation of the following registry parts (See Table5)
| ID | Part Name | Resistance | Description |
|---|---|---|---|
| 2-2O | <bbpart>J63003</bbpart> | A | yeast Kozak sequence |
| 3-11I | <bbpart>K105027</bbpart> | A | 'cyc100' minimal promoter |
August 24 (Tue)
- Colony check of part transformed yesterday: both 2-2O & 3-11I produced >100 colonies
- transfer to solution culture
- Miniprep of 2-4A, F1, F2, F3 followed by 'cut check' with EcoRI, SpeI
- (RESULTS)
- Transfer of F1 to solution culture (why?)
- Preparation of glycerol stock of cell culture containing 001 (why?)
- 200ml of culture solution mixed with 100ml of 50% glycerol
- stored at -80°C
August 25 (Wed)
- Miniprep of parts in solution culture: 2-2O, 3-11I, F1
- Cut check of 3-11I & F1 with EcoRI, SpeI
- (RESULTS?)
August 26 (Thu)
- Transformation of <bbpart>BBa_K204022</bbpart>, <bbpart>BBa_K204025</bbpart>, and <bbpart>BBa_K204040</bbpart> to send to Shanghai ECUST team in China.
August 27 (Fri)
- Transfer of yesterday's transformed parts (all produced colonies) to solution culture
- Transformation of the following parts (See Table 6)
- using competent cells opened on 8/20
- Preparation of YPD yeast culture medium with the following recipe (See Table 7)
- pH was adjusted to 5.8
- autoclaved before use
- 12.5g (2.5%) agar added to 500ml and 21 YPD agar plates were prepared
- Preparation of 41 LB agar plates from pre-mixed broth powder and 1.5% agar
| ID | Part Name | Resistance | Description |
|---|---|---|---|
| 1-1K | <bbpart>BBa_J63010</bbpart> | A | Protein fusion vector (Silver standard) |
| 1-1I | <bbpart>BBa_J63009</bbpart> | A | Low copy protein fusion vector (Silver standard) |
| 3-3G | <bbpart>BBa_K157013</bbpart> | A | 15aa glycine-serine linker (Freiburg standard) |
| MiliQ water | 1 liter | |
| Bacto tryptone | 20.0g | 2% |
| Bacto yeast extract | 10.0g | 1% |
| Glucose | 20.0g | 2% |
August 28 (Sat)
- Miniprep of parts in solution culture
- Restriction digest (for cut check) - 37°C for 30min
- 2-4A & 3-11I with EcoRI, SpeI
- 2-2O with XbaI, PstI
- K204022, K204025, K204040 wih EcoRI, PstI
- Gel electrophoresis of digests
- Plasmids not detected for 2-4A & 3-11I - mistake during miniprep? culture duration too long, plasmid loss occurred?
- Transfer of the following parts to solution culture
- 1-1K, 1-1I, 3-3G (yesterday's transformations)
- 2-4A, 3-11I (repeat pick-up from 10/22, 10/23 plates)
