Part:BBa_K1139022:Experience
PlacIq-M13-Plac-GFP on pSB3
Materials and Methods
1. Plasmid construction
pSB3K3- PlacIq-M13-Plac-GFP (BBa_K1139020)
pSB3K3-M13-Plac-GFP (BBa_K1139022)
2. Strain
DH5alpha (Ecoli of high competence)
JM109 (F+ strain Ecoli)
3. Media
Mix everything together in 1,000 mL autoclaved Elix H2O
LB
Bacto tryptone | 10 g/L |
Yeast Extract | 5 g/L |
NaCl | 10 g/L |
YT plate
Bacto tryptone | 8 g/L |
Yeast Extract | 5 g/L |
NaCl | 5 g/L |
Agarose | 15 g/L |
YT soft agar
Bacto tryptone | 8 g/L |
Yeast Extract | 5 g/L |
NaCl | 5 g/L |
Agarose | 6 g/L |
4. Protocol
- Preparation
1.Transform DH5alpha with pSB3K3- PlacIq-M13.
2.Grow overnight culture of the transformed DH5alpha and JM109 at 37°C.
- Plaque formation
3.Spin the overnight culture of the transformed DH5alpha at 9,000g for 1 minute.
4.Pipette the supernatant into a 1.5 mL tube.
5.Dilute it 100 times with water. (=> phage-particle-solution)
6.Melt YT soft agar using a microwave.
7.Dispense 3.5 mL of the melted soft agar to a new round tube, and keep them at 50°C in an incubator.
8.Dispense 400 µL of overnight culture of JM109 to a 1.5 mL tube.
9.Into the 1.5 mL tube, add 100 µL of the phage-particle-solution.
10.Transfer all the content of the 1.5 mL tube to the dispensed soft agar, mix them, and decant all of it on a YT plate.
Result
The result of the plaque forming assay is showed in Fig. 2. M13 phage genes and M13 origin worked together with lacIq promoter and pSB origin.
Fig. 2-A.
The plaques were formed using JM109 overnight culture and phage-particle-solution. The phage particles were obtained from the supernatant of the overnight culture of transformed DH5alpha (with pSB3K3- PlacIq-M13) at 9,000g. Mixture of 3.5 mL YT soft agar, 100 µL of X100 supernatant and 400 µL of overnight culture of JM109 was poured on a YT plate.
Fig. 2-B.
A mixture of only 3.5 mL YT soft agar and 500 µL of overnight culture of JM109 was poured on a YT plate.
For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/pSB-M13_Plasmid_Assay our work in Tokyo_Tech 2013 wiki].
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