Team:Uppsala-SwedenWeek13

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(→Identification of biobricks)
(→Construction Of J1,J2,J3)
 
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== Identification of biobricks ==
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== Construction Of J1,J2,J3 ==
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Plasmid of the samples which showed right size band on the gel were sent to a plasmid concentration test. We decided only J1, J2, J3 were in good concentration to proceed.
 
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However, we got the correct PCR lengths for Bio-bricks and results are shown in image files below.  
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Plasmid construction and verification of lengths for the last two levels, use of specific restriction sites to re-verify the constructs. Colonies obtained in the cells from the previous step were picked from the plate. Each colony was placed in 10ul of LB
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.The result was showed in Fig.1 and Fig2
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                              J1-J3
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[[Image:20100827 J1-J3 c-PCR 20100826 2 thumb.jpg|500px|center|border|Figure 1]]
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                              J2-J3
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[[Image:20100827 J2-J3 c-PCR 20100826 2 thumb.jpg|500px|center|border|Figure 1]]
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biobricks
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1ul of this LB was used to perform colony PCR.
-
However, we got the correct band lengths for all J1, J2, J3. And the one that are with correct band lengths were  
+
The product that obtained after running colony PCR were run on gel and plasmids lengths were verified. Colonies with correct plasmid lengths were selected and inoculated using the remaining 9ul of LB. Plasmid of the samples which showed right size band on the gel were sent to a plasmid concentration test. We decided only J1, J2, J3 were in good concentration to proceed.  
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selected.  
+
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The correct length samples of J1, J2, J3 were again inoculated again to get the enough plasmid for further progress.
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Second colony PCR was performed again for all the starting biobricks and the products were run for gel again. Comparing the DNA length to the band size, all the biobricks were confirmed.  
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The inoclum obtained from the overnight culture was used for plasmid extraction. The extracted plasmids were cut at specific restriction sites present in the biobrick sequence and run on the gel.
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The concentration of plasmid were measured for all the samples.
+
However, we got the correct PCR lengths for Bio-bricks and results are shown in image files below.
 +
The result was showed in Fig.1 and Fig2
 +
 +
                             
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[[Image:20100827 J1-J3 c-PCR 20100826 2 thumb.jpg|500px|center|border|thumb|J1-J3 Figure 1]]
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[[Image:20100827 J2-J3 c-PCR 20100826 2 thumb.jpg|500px|center|border|thumb|J2-J3 Figure 2]]
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Plasmid concentration of all starting biobricks.Plasmid of J1, J2, J3 were digested with proper enzymes. The biobricks were ligated in pairs according to the plan by Quick Ligase kit from Fermentas.
 
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The ligation product were transformed into DH5α competent cells and each sample were plated on the agar plate which antibiotics matched to its backbone antibiotic resistance.
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We got the correct band lengths for all J1, J2, J3. And the one that are with correct band lengths were
 +
selected. The correct length samples of J1, J2, J3 were again inoculated again to get the enough plasmid for further progress.

Latest revision as of 20:34, 26 October 2010

Week-13

Construction Of J1,J2,J3

Plasmid construction and verification of lengths for the last two levels, use of specific restriction sites to re-verify the constructs. Colonies obtained in the cells from the previous step were picked from the plate. Each colony was placed in 10ul of LB

1ul of this LB was used to perform colony PCR.

The product that obtained after running colony PCR were run on gel and plasmids lengths were verified. Colonies with correct plasmid lengths were selected and inoculated using the remaining 9ul of LB. Plasmid of the samples which showed right size band on the gel were sent to a plasmid concentration test. We decided only J1, J2, J3 were in good concentration to proceed.

The inoclum obtained from the overnight culture was used for plasmid extraction. The extracted plasmids were cut at specific restriction sites present in the biobrick sequence and run on the gel.

However, we got the correct PCR lengths for Bio-bricks and results are shown in image files below. The result was showed in Fig.1 and Fig2


J1-J3 Figure 1


J2-J3 Figure 2


We got the correct band lengths for all J1, J2, J3. And the one that are with correct band lengths were selected. The correct length samples of J1, J2, J3 were again inoculated again to get the enough plasmid for further progress.

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