Team:Cambridge/LabBook/Week7

From 2010.igem.org

(Difference between revisions)
Line 571: Line 571:
| 20µl
| 20µl
| 20µl H20
| 20µl H20
 +
|}
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===61. Experiment: Diagnostic gel of Gibson assembly reaction (Bill and Anja)===
 +
====Gel electrophoresis====
 +
Gel loading mixtures:
 +
{| class="wikitable"
 +
|-
 +
|
 +
|pA
 +
|B
 +
|pC
 +
|D
 +
|pE
 +
|G1
 +
|G2
 +
|G3
 +
|-
 +
|6x orange LD (µl)
 +
|3
 +
|3
 +
|3
 +
|3
 +
|3
 +
|3
 +
|3
 +
|3
 +
|-
 +
|5
 +
|2
 +
|5
 +
|5
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|1
 +
|12
 +
|12
 +
|12
 +
|-
 +
|12
 +
|15
 +
|12
 +
|12
 +
|16
 +
|5
 +
|5
 +
|5
 +
|}
 +
 +
Loaded gel:
 +
{| class="wikitable"
 +
|-
 +
|Easyladder 2
 +
|pA
 +
|B
 +
|pC
 +
|D
 +
|pE
 +
|G1
 +
|G2
 +
|G3
 +
|-
 +
|10µl
 +
|20
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|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:
 +
{| class="wikitable"
 +
|-
 +
|15µl
 +
|Gibson 1.33x Master Mix
 +
|-
 +
|2.5µl
 +
|pC (AB)
 +
|-
 +
|2.5µl
 +
|D (EG)
 +
|-
 +
|20µl
 +
|
|}
|}

Revision as of 16:22, 17 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.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
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