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Team:Cambridge/LabBook/Week7
From 2010.igem.org
Monday
51. Experiment: Transformation with pJS555 & pSB1C3 (Theo and Hannah)
Received a new plasmid from Jim Slock, so have transformed some chemically competent cells with it to build up a stock and test it later/we think it will glow for longer than the previous one (says Mr. Lovely)).
Theo transformed TOP10 with pSB1C3 from registry.
52. Experiment: Check for promoter+rbs+p.p.luc - obtain from the colony from transformation on page 37. (Paul)
Colony PCR
Take sample from colony with loop and put in 20μl of water to obtain DNA template.
Then prepare on isoFreeze PCR chiller in lister order:
- 10μl of 2xPhusion Master Mix
- 1μl of VR
- 1μl of VF2
- 1μl of DNA template
- 7μl of Nuclease-Free H20
Then PCR:
1. 98°C for 15 mins
2. 98°C for 10 secs
3. 65°C for 30 secs
4. 72°C for 1 min
5. repeat 2-4 35 times
6. 72°C for 10 mins
7. 4°C forever
Results:
Tube 1: 197.4ng/μl
Tube 2: 235.3ng/μl
Gel Electrophoresis
- Tube 1: 194.7ng/μl
- 4μl of DNA
- 3μl of 6x Orange Loading Dye
- 13μl Nuclease Free H20
- Tube 2: 235.3ng/μl
- 3μl of DNA
- 3μl of 6x Orange Loading Dye
- 14μl of Nuclease Free H20
53. Experiment: Extracting glycerol stocks of TOP10 with promoter+rbs+p.p.luc from registry. (Emily and Peter)
1. Take tube of glycerol stock from -80°C 2. Use loop/tooth pick (we used a loop) to streak on ampicillin resistance plate. Let it melt first, then streak.
NB NEVER let glycerol stocks thaw. They should be out and back in the -80°C freezer in 2-3mins max
3. Place in 30°C incubator overnight.
To be continued tomorrow.
54. Check for promoter+rbs+luc from p32 by luciferin injection. (Paul and Ben)
We used protocol described on p22.
3x Freezymes at -80°C, thawing at 37°C
55. Experiment: Gibson assembly of pBAD luxCDABEG in psB1C3
| PCR Construct | Forward Primer | Tm | Reverse Primer | Temp | Temp to use | Template DNA | Amplifies |
|---|---|---|---|---|---|---|---|
| Prefix.Pbad.Start of C | 9.prefix.f .pBadstart | 53 | 1.luxCstart.r .pBadend | 66.25 | 56 | Pbad | pBAD |
| End of Pbad.C.D.Start of A | 2.pBADend.f .luxCstart | 51.76 | 4.luxAstart.r .luxDend | 50.81 | 53 | Phk555 | CD |
| End of D.A.BStart of E | 3.luxDend.f .luxAstart | 54.32 | 6.luxEstart.r .luxBend | 71.56 | 57 | Phk555 | AB |
| End of B.E.G.Suffix | 5.luxBend.f .luxEstart | 51.63 | 7.suffix.r ev.luxGend | 56 | 54 | Phk555 | EG |
| End of G.Suffix.Plasmid.Prefix.Start of B | 8.luxGend.f or.suffix | 68.73 | 10.pBadstart.r .prefix | 72 | 71 | Plasmid Backbone (BBa_Jo4450) | pSB1C3 |
Added (on ice!) in 5 individual (A-E) PCR tubes
| 2x Phusion Master Mix | 25µl |
| Forward Primer | 0.25µl |
| Reverse Primer | 0.25µl |
| Template DNA | 2µl |
| Distilled water | 22.5 |
| 50µl |
|---|
Programs on PCR machines
| start | cycle | machine 1 | machine 2 | machine 3 | end | ||
|---|---|---|---|---|---|---|---|
| 98°C | 98°C | 56°C | 53°C | 71°C | 72°C | 72°C | 10°C |
| 30s | 10s | 15s | 15s | 15s | 1.45min | 7.5min | ad infinitum |
| A&C | B&D | E | |||||
| Denaturation | Annealing | Elongation | |||||
| 30x |
Results: Machine 1 had a power fail very close to the end of the program Machine 2 had a loose lid; the tubes popped open and there was no liquid left Machine 3 had entirely melted the top of the PCR tube
The experiment was repeated, but this time all reactions (A-E) were run in Machine 2 (see p.38 for program).
Nanodrop Measurements
| A | pBAD | 455.6ng/µl |
| B | CD | 339.3ng/µl |
| C | AB | 937.4ng/µl |
| D | EG | 165.1ng/µl |
| E | pSb1C3 | 430.8ng/µl |
| pA | pBAD | 173.5ng/µl |
| pC | AB | 159.7ng/µl |
| pE | 974.6ng/µl |
blanked with distilled water
Gel electrophoresis
Gel loading mixtures were made up according to:
| A | B | C | D | E | pA | pC | pE | |
| 6x orange LD (µl) | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
| plasmid DNA (µl) | 2 | 2 | 1 | 5 | 2 | 5 | 5 | 1 |
| nuclease-free water (µl) | 15 | 15 | 16 | 12 | 15 | 12 | 12 | 16 |
final volume 20µl
A 1% agarose E-gel was loaded as follows
| Easyladder 2 | A | B | C | D | E | pA | pC | pE |
| 10µl | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 |
Bands were observed at the following lengths:
| A | B | C | D | E | pA | pC | pE |
| 1-2 | 3-5 | 2-3 | 2 | 2 | 1-2 | 2 | 1-2 |
Bands for pA, B, pC, D & pE were cut out the gel and DNA was extracted following the QIAquick Gel Extraction Kit protocol
Preparation of Gibson assembly 1.33x master mix
| Taq ligase 40u/µl | 50µl |
| 5x isothermal buffer | 100µl |
| T5 exonuclease 1u/µl | 2µl |
| Phusion polymerase 2u/µl | 6.25µl |
| Nuclease--free water | 216.75 |
| 375µl |
Gibson Assembly Reaction added to PCR tube:
| 15µl | Gibson 1.33x master mix |
| 1µl | purified pA |
| 1µl | purified B |
| 1µl | purified pC |
| 1µl | purified D |
| 1µl | purified pE |
| 20µl |
Incubated for 1h at 50C Stored at 4C overnight
| length | nanodrop after gel | ||
| pBAD | 1210b | (A) | 22.7ng/µl |
| CD | 2388b | (B) | 2.7ng/µl |
| AB | 2097b | (C) | 10.0ng/µl |
| EG | 1863b | (D) | 20.9ng/µl |
| pSB1C3 | 2072b | (E) | 5.1ng/µl |
Tuesday
56. Experiment: Transformation of TOP10, red strain and with Gibson assembly (pBAD, luxCDABEG, pSB1C3) (Will and Anja)
Followed protocol on p13+14. Transformed:
| with Gibson reactions | |
| TOP10 | 1, 2, 3 |
| red (BW25113 Δhns::kan) | 1, 2, 3 |
| black (GM230 hns-205::Tn10 TetR) | 1, 2, 3 |
Plated 150µl on LB agar plates with Chl. each (9pl.)
57. Experiment: Repeat gel electrophoresis from p.39+40 (Will and Anja)
Followed protocol on p.39+40 (pcr products had been kept in -20°C overnight).
Results: A, C, D, pA & pC were of appropriate size. B (again 3-5kb) and E, pE (slightly lower than expected, closer to D than C) were of inappropriate size.
It was decided to run new PCR reactions for B (CD) and E (pSB1C3) following the protocol on p.38.
Nanodrop measurements: (blanked with Di H20)
B (CD) 980.7ng/µl
E (pSB1C3) 127.5ng/µl
Gel Electrophoresis:
Gel loading mixtures made up as follows:
| E | B | |
| 6x Orange LD (µl) | 3 | 3 |
| plasmid DNA (µl) | 7 | 1 |
| nuclease free H20 (µl) | 10 | 16 |
A 1% Agarose (E-gel) was loaded as follows:
| Easyladder 2 | E | B | all other wells filled H20 |
| 10µl | 20µl | 20µl |
58. Experiment: Making liquid culture of tetR repressed promoter, rbs + luc in TOP10 (from registry) (Peter and Emily)
Make LB broth with campicillin, adding 200µl ampicillin to 200ml LB to make 100µg/ml concentration final solution.
Plates prepared from glycerol stocks had grown well, but were transferred from 30°C to 37°C incubator for better growth - the temperature effect on luciferase won't matter. The coonies were very small. A single colony was chosen using a loop, placed in 3ml of LB&Amp and shaken. 3 tubes were used and placed in the 30°C incubator overnight.
59. Experiment: Gel Extraction (Anja)
Results from Gel electrophoresis on p.42.
E showed a band slightly above 2kb.
B again showed a band between 3-5kb.
Bands for A & pA, B, pC, D, pE were cut out from 'repeated' gel on p.41 and 'Enew' as well as a trace of seemingly correctly sized B ('B trace') were cut out from gel on p.42. DNA was extracted following the 'QIAquick Gel Extraction Kit (miniElute columns)' protocol.
The purified DNA was stored at 4°C overnight.
Wednesday
Nanodrop measurements:
| A | 38.8ng/µl |
| B | 24.7ng/µl |
| B trace | 32.5ng/µl |
| C | 13.2ng/µl |
| D | 24.0ng/µl |
| E | 14.5ng/µl |
| E new | 13.3ng/µl |
60. Experiment: PCR of B trace with primers for amplification of B & gel electrophoresis (Will and Anja)
Followed protocol on p.38.
Nanodrop measurements: B trace PCR 492.2ng/µl
Gel Electrophoresis
Gel loading mix:
| 6x orange LD | 3µl |
| plasmid DNA | 2µl (B Trace PCR) |
| DI H20 | 15µl (nuclease-free) |
Gel loading as follows:
| Easyladder 2 | 3 trae PCR | all other wells filled with |
| 10µl | 20µl | 20µl H20 |
