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Team:Cambridge/LabBook/Week6
From 2010.igem.org
42. Experiment: Transforming TOP10 with pBAD promoter (Hannah)
Took plasmid from Shuna, followed protocol from earlier, growing up stock to make more plasmid so we can PCR from it later.
Plated out on Kanomycin plates
43. Experiment: Plasmid extraction - pHK555 + LuxR from registry (Hannah)
Followed Qiagen miniprep protocol
| Plasmid | Concentration (ng/μl) |
|---|---|
| pHK555 | 20.9 |
| 17.3 | |
| LuxR | 43.9 |
| 89.2 |
These are in storage in the -20°C
44. Experiment: Growth of Slock10, TOP10 pHK555+pHK724 and phosphoreum to make glycerol stocks for tomorrow (Hannah)
- Used antibiotic resistance markers to select for bacteria in Slock10 + TOP10
- These were put in the 37°C incubator
45. Experiment: Growing up of 10ml overnight cultures of old glowth (from non-glowing flask) and BBa_216007 (Peter)
Made 4 vials:
- Amp + Chl glowth?
- Amp glowth?
- Amp glowth? + lumazine (BBa_216007), double inoculation
- Amp lumazine
Placed in incubator at 30°C
46. Experiment: Transformation of TOP10cc with BBa_R0040 (TetR repressed promoter), BBa_E0030 (enhanced yellow fluorescent protein) separately. (Theo & Anja)
BBa_R0040 (Plate 1, Well 6L) and BBa_E0030 (Plate 2, Well 24E) were resuspended in 10μl deionised H20.
TOP10cc were taken out of -80°C freezer and thawed on ice. 1ml pipette tip cut with scissors (sterile). 2x50μl of TOP10cc were transformed to 1.5ml eppendorf tubes.
2μl of resuspended BBa_R0040 and 2μl of resuspended BBa_E0030 were added to separate eppendorf tubes with TOP10cc. Cells were held on ice for 30mins. Heat shocked for 60s at 42°C (waterbath). Put on ice for ~2mins. Added 250μl pre-warmed (37°C) soc. Incubated at 37°C for 1h with rotation. Plated 50μl on pre-warmed (in 37°C incubator) LB agar plates with:
- Amp for BBa_R0040
- Kan for BBa_E0030
Grow colonies overnight at 37°C.
46. Experiment: Streaking out and setting up overnight cultures of TOP10 with promoter + rbs + luc (Anja)
Streaked out from a single colony on an LB agar plate with Amp and incubate at 37°C overnight.
Inoculated single bacterial colony in 5ml LB with 5μl (100mg/ml) Ampicillin, incubated at 37°C with shaking overnight.
47. Experiment: Using plate reader to characterise light output of the pHK724+pHK555 in Slock10 and TOP10 as they grew. (Paul)
Grew the following (1/2 Slock10, 1/2 TOP10):
- 10 x Cells + Medium LB + Amp
- 10 x Cells + Medium LB + Chl
- 10 x Cells + Medium LB + Amp + Chl
Final concentrations of Amp = 100μg/ml, Chl = 20μg/ml
Growth - at 30°C as we hope this will maximise light output.
Volume per well - 100μl of total (= LB + Chl/Amp + Cells)
Cells used - grown up in broth on previous day by Paul in LB + antibiotic broth.
48. Experiment: Making long-term stock of TOP10 with promoter + rbs + p.p.luc (Peter)
Added 0.5ml 40% glycerol solution and 0.5ml sample culture to cryogenic vial (x2). Gently vortexed.
Stored in -80°C freezer in 'Black-GM230hns-205' storage box. Vials labelled with sticker 'TetR luc'.
49. Experiment: Transfer of promoter + rbs + p.p.luc into psB1C3 for submission to registry. (Will, Peter & Anja)
1.5ml of TOP10 cells with promoter+rbs+luc grown overnight in LB+Amp were transferred to eppendorf tubes (x2) and cells were spun down in microcentrifuge (1min, 13000rpm). Suernatant was discarded and plasmid was purified following the 'QIAprep Spin Miniprep Kit using a microcentrifuge' protocol.
Nanodrops showed 135.1ng/μl DNA present in purified plasmid prep (EB as blank).
Restriction enzyme digest
Prepared at room temperature in the listed order:
| nuclease-free H20 | 14μl |
| 10x Fast Digest Buffer | 2μl |
| Plasmid DNA | 2μl |
| FD Enzyme EcoRI | 1μl |
| FD Enzyme PstI | 1μl |
| 20μl |
It was checked (Nanodrop!) that 2μl of plasmid DNA would not contain more than 1μg of DNA. The reaction was mixed gently, spun down and incubated at 37°C (PCR machine) for 30mins. Enzymes were inactivated by heating at 80°C for 10mins (in PCR Machine).
DNA Purification
was carried out following the 'QIAquick PCR Purification Kit Protocol (using a microcentrifuge)' The DNA was stored overnight at -20°C.
===50. Experiment: Continued transfer of promoter + rbs + p.p.luc into psB1C3 for submission to registry. (Will and Anja)
Gel Electrophoresis
Nanodrop reading was taken for the restriction enzyme digest.
Plasmid and promoter + rbs + luc: 1ng/μl
1% Agarose gels were poured following the below protocol:
Preparation 1% Agarose Gels (2 gels)
1g (agarose)/100ml (TAE - better resolution > 4kb / TBE - better resolution 0.1-3kb) --> 0.5 - 10kb optimal DNA resolution
- Measure agarose into beaker with buffer (50ml for 7x10cm gel box)
- Microwave until agarose fully melted
- Let agarose cool on bench
- Seal open edges of gel box with tape
- Add 10μl Sybr Safe Dye to agarose sultion, swirl
- Pour agarose solution in taped gel box, remove bubbles, put in comb, letit cool & solidify
Due to the low DNA concentration, it was decided to repeat the restriction enzyme digest (x3) following the same protocol as on p.31 without the enzyme inativation step.
Nanodrop measurements were taken for the restriction enzyme digests:
| Plasmid and promoter + rbs + luc 1 | 34.9ng/μl |
| Plasmid and promoter + rbs + luc 2 | 14.4ng/μl |
| Plasmid and promoter + rbs + luc 3 | 35.8ng/μl |
Gel loading mixture was mude up: (4x) 3μl 6x orange loading dye 7μl plasmid DNA (restriction digest) --> 245ng DNA 10μl deionised H20 20μl
Gel was loaded as follows:
| Easyladder II | Plasmid & promoter + rbs + luc (4 wells) |
| 10μl | 20μl |
Gel was run for 40mins at 100V. No bands were observed.
Therefore, the restriction enzyme digest was repeated as on p.32.
Nanodrop readings:
| Plasmid and promoter + rbs + luc 1 | 14.6ng/μl |
| Plasmid and promoter + rbs + luc 2 | 43.8ng/μl |
| Plasmid and promoter + rbs + luc 3 | 18.1ng/μl |
4 gel loading mixtures were taken up.
| Digest 1 | Digest 2a | Digest 2b | Digest 3 | |
| 6x orange LD (μl) | 3 | 3 | 3 | 3 |
| Plasmid DNA (μl) | 4 | 10 | 5 | 5 |
| Deionised H20 (μl) | 13 | 7 | 12 | 12 |
| 58.4ng DNA | 438ng DNA | 219ng DNA | 90ng DNA |
Gel was loaded as follows:
| Easyladder II | Digest 1 | Digest 2a | Digest 2b | Digest 3 |
| 10μl | 20μl | 20μl | 20μl | 20μl |
Empty wells were loaded with deionised H20. The gel was run and two bands were observed. One just around 2kb --> plasmid backbone (2079b) and one a little lighter --> promoter+rbs+luc (1727b). The lower band was cut out of the gel and DNA was extracted following the QIAquick Gel Extraction Kit Protocol.
