Team:Cambridge/LabBook/Week7

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===55. Experiment: Gibson assembly of  pBAD luxCDABEG in psB1C3===
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Revision as of 10:39, 7 September 2010

Contents

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.rev.luxGend 56 54 Phk555 EG
End of G.Suffix.Plasmid.Prefix.Start of B 8.luxGend.for.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