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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 trace PCR | all other wells filled with |
| 10µl | 20µl | 20µl H20 |
61. Experiment: Diagnostic gel of Gibson assembly reaction (Bill and Anja)
Gel electrophoresis
Gel loading mixtures:
| pA | B | pC | D | pE | G1 | G2 | G3 | |
| 6x orange LD (µl) | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
| 5 | 2 | 5 | 5 | 1 | 12 | 12 | 12 | |
| 12 | 15 | 12 | 12 | 16 | 5 | 5 | 5 |
Loaded gel:
| Easyladder 2 | pA | B | pC | D | pE | G1 | G2 | G3 |
| 10µl | 20 | 20 | 20 | 20 | 20 | 20 | 20 | 20 |
Result: In neither G1, G2 or G3 did any high molecular weight bands occur other than those for pA, B, pC, D and pE individually => Gibson assembly reaction did not work.
62. Experiment: Test Gibson Master Mix for functionality (Bill and Anja)
Gibson assembly reaction
Added to a PCR tube:
| 15µl | Gibson 1.33x Master Mix |
| 2.5µl | pC (AB) |
| 2.5µl | D (EG) |
| 20µl |
Incubated for 1h at 50°C
Gel electrophoresis
| pC | D | G1 | G2 | |
| 6x orange LD (µl) | 3 | 3 | 3 | 3 |
| plasmid DNA (µl) | 5 | 5 | 17 | 17 |
| nuclease-free H20 | 12 | 12 | - | - |
(All other wells were filled with DI H20)
Gel loaded as follows:
| Easyladder 2 | pC | D | G1 | G2 |
| 10µl | 20µl | 20µl | 20µl | 20µl |
| Results: | ~3kb | 2-3kb | 1 over 5kb, | 1 over 5kb, |
| 1 around 5kb | 1 around 3-5kb | |||
| After 10 more mins: | ~2.3kb | ~1.9kb | 5kb, 2.5kb, 2.1kb | 5kb, 2.5kb, 2.1kb |
=> Gibson Master Mix is functional
Thursday
63. Experiment: Gibson assembly of pBAD, luxCD AB EG and pSB1C3 as well as all individual 'neighbour parts' in pairs (Will and Anja)
| Gibson Master Mix | A (pBAD) | B (CD) | C (AB) | D (EG) | E (pSB1C3) | |
| G1 (µl) | 15 | 1 | 1 | 1 | 1 | 1 |
| G2 (µl) | 15 | 2.5 | 2.5 | |||
| G3 (µl) | 15 | 2.5 | 2.5 | |||
| G4 (µl) | 15 | 2.5 | 2.5 | |||
| G5 (µl) | 15 | 2.5 | 2.5 | |||
| G6 (µl) | 15 | 2.5 | 2.5 | |||
| j1 (µl) | 15 | 1 | 1 | 1 | 1 | 1 |
| j2 (µl) | 15 | 2.5 | 2.5 | |||
| j3 (µl) | 15 | 2.5 | 2.5 | |||
| j4 (µl) | 15 | 2.5 | 2.5 | |||
| j5 (µl) | 15 | 2.5 | 2.5 | |||
| j6 (µl) | 15 | 2.5 | 2.5 |
G1-G6: A->E (new) refer to gel extracted PCR fragments
j1-j6: A->E (pA, B, pC, D & pE) refer to PCR reactions (that have not been run on a gel and gel extracted)
Gibson assembly reactions were made up in PCR tubes according to the table above. The reaction mixtures were spun down (mixed by flicking tube), and incubated at 50°C.
Gel Electrophoresis
2 separate gels were run:
1. Gel loading mixtures:
| pA | B | pC | E | j1 | j2 | j3 | j4 | j5 | j6 | |
| 6x Orange LD (µl) | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
| Plasmid DNA (µl) | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| Nuclease-free H20 (µl) | 12 | 12 | 12 | 12 | 12 | 12 | 12 | 12 | 12 | 12 |
Gel was loaded as follows:
| Easyladder II | pA | B | pC | E | j1 | j2 | j3 | j4 | j5 | j6 |
| 10µl | 20µl | 20µl | 20µl | 20µl | 20µl | 20µl | 20µl | 20µl | 20µl | 20µl |
2. Gel loading mixtures:
| A | B | C | Enew | G1 | G2 | G3 | G4 | G5 | G6 | |
| 6x Orange LD (µl) | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
| Plasmid DNA (µl) | 1 | 1 | 1 | 1 | 5 | 5 | 5 | 5 | 5 | 5 |
| Nuclease-free H20 (µl) | 16 | 16 | 16 | 16 | 12 | 12 | 12 | 12 | 12 | 12 |
Gel was loaded as follows:
| Easyladder II | A | B | C | E | G1 | G2 | G3 | G4 | G5 | G6 |
| 10µl | 20µl | 20µl | 20µl | 20µl | 20µl | 20µl | 20µl | 20µl | 20µl | 20µl |
Transformation of TOP10 with 1)G1 and 2)j1
Followed protocol on p13+14, however, we transformed with 10µl Gibson reaction instead of 2µl. Plated 150µl on LB agar plates with Chl. Incubated at 30°C overnight.
Result: All 4 plates blank after overnight :( All 4 growth after 2 days, RFP present, no glow.
Friday
Results: No colonies observed from above transformation.
64. Expt: Transfer of promoter+rbs+P.P.Luc into pSB1C3 for submission to registry (Emily + Anja)
Followed protocol on p31 (nanodrop:[plasmid]=26.5ng/µl)
Restriction enzyme digest
Prepated at RT in the following order:
| nuclease-free H20 | 1µl |
| 10x Fast Digest Buffer | 2µl |
| Plasmid DNA | 15µl |
| FD Enzyme EcoRI | 1µl |
| FD Enzyme PstI | 1µl |
| 26.5ng/µl |
The digest was prepared twice. It was checked that 15µl of plasmid DNA would not contain more than 1µg of DNA. The reaction was mixed gently, spun down and incubated at 37°C for 4h45min (in PCR machine).
Ligation
Nanodrop measurements: Digest 1 -> 18.3ng/µl Digest 2 -> 22.8ng/µl
pSB1C3 supplied at 25ng/µl. 10-100ng of linearised vector DNA should be added to ligation mix. 3µl give 75ng. 3:1 molar excess of insert DNA should be added.
75ng/2035b x 1727b x 3 = 190ng => 9µl
To a PCR tube add:
| promoter + rbs + luc digest 2 | 9µl |
| pSB1C3 | 3µl |
| 5x rapid ligation buffer | 4µl |
| T4 DNA ligase | 1µl |
| nuclease-free H20 | 3µl |
| 20µl |
The ligation was prepared twice. Mix was vortexed, spun down and incubated at 22°C for 1h45min (in PCR machine).
Transformation
Followed protocol on p13+14. Transformed 2x50µl TOP10cc with ligation 1 and ligation 2 (10µl of ligation rather than 2µl). Plated on LB agar plates with Chloramphenicol (plated 150µl).
Results: No colonies from TOP10 transformation with ligation. Plate 1 of TOP10 cells transformed with ligation 1: 2 colonies. Plate 2: 5 colonies.
65. Expt: Gibson assembly of pBAD, luxCDABEG (amplified in one fragment) and pSB1C3 (Will and Anja)
| Template | Forward Primer | Reverse Primer | Fragment Amplified |
| I0500 | prefix.f.pBadstart | luxCstart.r.pBadend | pBAD |
| pJS555 | pBADend.f.luxCstart | suffix.rev.luxGend | luxCDABEG |
| BBa_J04450 | luxGend.for.suffix | pBadstart.r.prefix | pSB1C3 |
(BBa_J04450 had one TOP10 colony in DI H20)
PCR mix made up as on p38.
Program of PCR machine:
For pBAD and luxCDABEG:
| 98°C | 30s | |
| Start cycle (30x) | 98°C | 10s |
| 55°C | 15s | |
| End cycle | 72°C | 3min |
| 72°C | 7.5min | |
| 10°C | forever |
For pSB1C3:
| 98°C | 30s | |
| Start cycle (30x) | 98°C | 10s |
| 71°C | 15s | |
| End cycle | 72°C | 1.45min |
| 72°C | 7.5min | |
| 10°C | forever |
Gel Electrophoresis
1% (self-cast) Agarose gel loaded as follows:
| Easyladder II | pBAD | luxCDABEG 1 | luxCDABEG 2 | pSB1C3 |
| 20µl | 25µl | 25µl | 25µl | 25µl |
+ 5µl 6x orange LD (mixed before adding to gel) to everything except the ladder.
run at 120V
Results: Only for pBAD did we observe a band (this was of correct size).
=> PCR of luxCDABEG from pJS555 failed
=> PCR of pSB1C3 from BBa_J04450 transformed colony failed.
Following the 'MinElute Gel Extraction' protocol pBAD was isolated (using MinElute columns) and stored at 4°C.
66. Expt: PCR of pJS555 colony PCR of cells containing pSB1C3 (&RFP biobrick), PCR of pBAD (Will & Hannah)
Templates:
1. pJS555 was diluted 10x in nuclease free water (taken from vial sent by James Slock), and PCRed in 3 fragments: CD, AB, EG using primers ordered on 17.08.10.
2. A colony grown up with a pSB1C3 plasmid (with RFP biobrick) was suspended in 20µl DI autoclaved water. 2µl of this was added to standard PCR mix as "template".
3. Purified I0500 (from Shuna)
Mixture was:
| 2x Phusion Master Mix | 27.5µl |
| Forward Primer | 0.25µl |
| Reverse Primer | 0.25µl |
| Template DNA | 2µl |
| DI (autoclaved) H20 | 25µl |
Naming system is as on pp38-41.
A, C, b, d in machine (2)
e in machine (3)
In addition, another b was run in machine (3) in an attempt to get rid of the problematic secondary structure in the template DNA (hypothesised). This tube was labelled B72.
After PCR tubes were run on Agarose on a single gel
Gel Preparation
- 150ml TAE + 1.5g Agarose + 10µl SYBERSAFE were prepared
- 8 Tooth comb was used
- each well could house 30µl liquid
