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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 |
