Team:Cambridge/LabBook/Week8

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Week 8: Monday 30th August - Sunday 5th September

Contents

Monday

Result (from Expt. 68):

No growth overnight, 2 growths out of 3 plates (+1 fungus) after weekend of growing. No glow.

69. Expt: Growing up of cell cultures of above experimental results & of results on p49 (Will)

Strains G1, j1 and G28wh were grown up in 5ml LB + 2µl Chloramphenicol, and on plates containing LB+Cm.

Left to grow at 30°C for 24h.

Results: Strains grew but did not glow after 48h.

70. Expt: Miniprep pSB1C3 from colonies (Anja)

Suspended a 'streak' of bacteria transformed with BBa_J04450 (registry part in pSB1C3) in 250µl buffer P1. Followed 'Bench protocol: Qiagen Spin Miniprep Kit Using a Microcentrifuge'.

Nanodrop 12.8ng/µl

71. Expt: Gibson assembly of pBAD, luxCD,AB,EG and pSB1C3 (Anja)

PCR:

Template Primer f. Primer r. Amplified Fragment
I0500 prefix.f.pBadstart luxCstart.r.pBADend pBAD (A)
pJS555 pBADend.f.luxCstart luxAstart.r.luxDend luxCD (B)
pJS555 luxDend.f.luxAstart luxEstart.r.luxBend luxAB (C)
pJS555 luxBend.f.luxEstart suffix.rev.luxGend luzEG (D)
BBa_J04450 luxGend.for.suffix pBADstart.r.prefix pSB1C3 (E)

BBa_J04450: once purified (E), once colony PCR (EC)

Followed protocol on p38 for PCR mixtures and programs.

Gel electrophoresis

Self-cast 1% agarose gel loaded as follows:

Easyladder II A A B B C C D D E E EC EC
10µl 17µl 17µl 17µl 17µl 17µl 17µl 17µl 17µl 17µl 17µl 17µl 17µl

Run at 120V.

No bands were visible. Not even in the marker lane. Since a gel was that had been sitting on the bench for a few days and SYBR Safe Dye is light sensitive it is assumed that the dye had become non-functional.

A 1% Agarose gel was loaded as follows:

Easyladder II A A B C D E? EC?
10µl 12µl 12µl 12µl 12µl 12µl 12µl 12µl PCR Rxn
3µl 3µl 3µl 3µl 3µl 3µl 3µl 6x LD
5µl 5µl 5µl 5µl 5µl 5µl 5µl Nuclease-free H20

These were all mixed and spun down prior to gel loading.

Bands of correct sizes were observed in all cases.

Gel Extraction

DNA was extracted from the gel following the "Bench protocol: MinElute Gel Extraction Microcentrifuge protocol".

Tuesday

72. Expt: Continued Gibson assembly of pBAD, luxCD,AB,EG and pSB1C3 (Anja)

Fresh Gibson 1.33x Master Mix was prepared following the protocol on p40.

A Gibson Assembly reaction was prepared according to the protocol on p40, but taking twice the volume of the Master Mix (ie 30µl) and each fragment (ie 2µl). The reaction was incubated for 1h at 50°C.

Transformation

TOP10 cells, red strain and black strain were transformed with 10µl of Gibson reaction following protocol on p13+14. Plated 150µl on LB agar plates with Chl. (Since we were short of LB+Agar+Chl plates, two old LB agar + 5µg/ml Chl plates from PJ were used). Incubated at 30°C overnight.

73. Expt: Prepare DNA sequencing material for BioScience (Theo and Anja)

Extracted plasmids from TOP10 cells were transformed with pBAD, luxCD,AB,EG in pSB1C3 following the 'Qiaprep Spin Miniprep Kit Using a Microcentrifuge' protocol.

Experiment was cancelled (to be repeated on 01/09/10)

Wednesday

74. Expt: Prepare DNA sequencing material for BioScience (Anja)

Extracted plasmids from TOP10 cells were transformed with pBAD, luxCD,AB,EG in pSB1C3 following the 'Qiaprep Spin Miniprep Kit Using a Microcentrifuge' protocol.

Nanodrop measurements: 88.7ng/µl

Primers are at 100µM = 100pmol/µl --> 30x dilution --> 3.2pmol/µl

BioScience requests DNA concentrations of 100ng/µl for plasmids (Volume: 6µl) and 3.2pmol/µl (Volume: 10µl) for primers.

  • Tube 1: 6µl of TetR repr. prom, rbs, P.P.luc in pSB1C3 (purified) at 88.7ng/µl
  • Tube 2: 10µl Forward primer of 3.2pmol/µl (made up from 0.33µl 100pmol/µl primer and 9.67µl nuclease-free H20)
  • Tube 3: 10µl Reverse primer of 3.2pmol/µl (made up from 0.33µl 100pmol/µl primer and 9.67µl nuclease-free H20)

75. Expt: Repeat of PCR of pBAD+luxCDABEG using modified reaction conditions (Will)

Added on ice:

2x Phusion Master Mix 25µl
Forward primers 0.25µl
Reverse primers 0.25µl
Template DNA 2µl
Distilled (nuclease-free) H20 22.5µl

notation as on p38

Fragment Template Reaction
110°C heated lid, 1m30s denaturation
Promoter I0500 purified (James Brown) 35 cycles
CD pJS555 30s 98°C, 30s 53°C, 1m45s 72°C
AB pJS555 7m30 elongation
EG pJS555
pSB1C3 Plasmid extract from BBa_J04450 strain Same as above only 71°C

In addition EG was run in touchdown PCR.

Heated lid 112°C
Denaturation 98°C 1m30
Touchdown 70°C to 45°C 45 cycles
Elongation 72°C 5min

PCR products were loaded into 1g/100ml agarose gel with 20µl/100ml SYBR SAFE strain.

Per well 25µl product + 5µl Gel loading dye

Wells: Hyperladder I, A, B, C, D, E, Dtouchdown, Hyperladder I

76. Night of 1 September - Plate Reader

In two parts:

(A)

Arabinose induction of G28 (lux operon under pBAD)

Rows A and B: all LB+Chl (100µl)

Column Inoculated with Arabinose Conc x val
1 G28 0 x1, 11
2 G28 1µM x2, 12
3 G28 10µM x3, 13
4 G28 100µM x4, 14
5 G28 1mM x5, 15
6 G28 100mM x6, 16
7 pSB1C3 0 x7, 17
8 Nothing - LB only 0 x8, 18
9 Slock10 0 x9, 19
10 Empty well x10, 20

Incubated at 30°C

Plate reader protocol:

  • Lum reads: gain 3200, time = 10s
  • OD reads at 100µl (595nm)
  • 10 mins between readings

Layout:

1 8
D x21 x28
E x33 x40