Difference between revisions of "Part:BBa K1139020:Experience"

 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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<partinfo>BBa_K1139020</partinfo>
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how you used this part and how it worked out.
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===1. Materials and Method===
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'''1-0 Plasmid construction'''<br>
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pSB3K3- PlacI<sup>q</sup>-M13-Plac-GFP (<partinfo>BBa_K1139020</partinfo>)<br>
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pSB3K3-M13-Plac-GFP (<partinfo>BBa_K1139022</partinfo>)<br>
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[[Image:Titech2013_parts_K1139020_EX_Fig1.jpg|thumb|left|500px|'''Fig. 1.''' Plasmid construction for assay]]
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<br><br><br><br><br><br><br><br><br><br>
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'''1-1 Strain'''<br>
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DH5alpha (''Ecoli'' of high competence)<br>
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JM109 (F+ strain ''Ecoli'')<br>
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'''1-2 Media'''<br>
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Mix everything together in 1000 mL autoclaved Elix H<small>2</small>O<br>
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LB<br>
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[[Image:Titech2013_parts_K1139020_EX_Tab1.jpg|frameless|left|500px|]]
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<br><br><br><br>
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YT soft agar<br>
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[[Image:Titech2013_parts_K1139020_EX_Tab2.jpg|frameless|left|500px|]]
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<br><br><br><br>
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YT plate<br>
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[[Image:Titech2013_parts_K1139020_EX_Tab3.jpg|frameless|left|500px|]]
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<br><br><br><br>
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'''1-3Protocol'''<br>
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*Preparation<br>
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1.Transform DH5alpha with pSB3K3- PlacI<sup>q</sup>-M13.<br>
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2.Grow overnight culture of the transformed DH5alpha and JM109 at 37°C.<br>
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*Plaque formation
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3.Spin the overnight culture of the transformed DH5alpha at 9,000g for 1 minute.<br>
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4.Pipette the supernatant into a 1.5 mL tube.<br>
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5.Dilute it 100 times with water.  (=>phage-particle-solution)<br>
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6.Melt YT soft agar using a microwave.<br>
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7.Dispense 3.5 mL of the melted soft agar to a new round tube, and keep them at 50°C in an incubator.<br>
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8.Dispense 400 µL of overnight culture of JM109 to a 1.5 mL tube.<br>
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9.Into the 1.5 mL tube, add 100 µL of the phage-particle-solution.<br>
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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.<br>
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===2. Result===
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The result of the plaque forming assay is showed in Fig. 2.  M13 phage genes and M13 origin worked together with ''lacI<sup>q</sup>'' promoter and pSB origin.<br>
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[[Image:Titech2013_parts_K1139020_Fig1.jpg|thumb|left|500px|'''Fig. 2-A.''' The plaques]]
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[[Image:Titech2013_parts_K1139020_Fig2.jpg|thumb|left|500px|'''Fig. 2-B.'''  The negative control]]
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<br><br><br><br><br><br><br><br><br><br><br><br>
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'''Fig. 2-A.'''<br>
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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- PlacI<sup>q</sup>-M13) at 9,000g.  Mixture of 3.5 mL YT soft agar, 100 µL of X 100 supernatant and 400 µL of overnight culture of JM109 was poured on a YT plate.<br>
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'''Fig. 2-B.'''<br>
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A mixture of only 3.5 mL YT soft agar and 500 microL of overnight culture of JM109 was poured on a YT plate.<br>
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===Applications of BBa_K1139020===
 
===Applications of BBa_K1139020===

Revision as of 08:43, 26 September 2013

BBa_K1139020

1. Materials and Method

1-0 Plasmid construction
pSB3K3- PlacIq-M13-Plac-GFP (BBa_K1139020)
pSB3K3-M13-Plac-GFP (BBa_K1139022)

Fig. 1. Plasmid construction for assay











1-1 Strain
DH5alpha (Ecoli of high competence)
JM109 (F+ strain Ecoli)

1-2 Media
Mix everything together in 1000 mL autoclaved Elix H2O
LB

Titech2013 parts K1139020 EX Tab1.jpg





YT soft agar

Titech2013 parts K1139020 EX Tab2.jpg





YT plate

Titech2013 parts K1139020 EX Tab3.jpg





1-3Protocol

  • 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.

2. 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
Fig. 2-B. The negative control













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 X 100 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 microL of overnight culture of JM109 was poured on a YT plate.


Applications of BBa_K1139020

User Reviews

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