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From 2010.igem.org

Week-9

Identification of Bio-bricks

Plasmid of the samples which showed right size band on the gel were sent to a plasmid concentration test. We decided only F2, F6, F7, F8, F15 and F16 were in good concentration to proceed.

Sample A[260] A[280] Bg[320] Ratio Protein (μg/ml) Nucleic Acid (μg/ml) f2 0,0946 0,0607 0,0202 f3 0,0444 0,0346 0,0216 1,7474 2,9830 0,9640 f6 0,0844 0,0669 0,0300 1,4721 16,1731 2,0934 f7 0,1348 0,1020 0,0557 1,7074 11,9965 3,3075 f8 0,0905 0,0690 0,0420 1,7961 5,1758 2,0767 f11 0,0332 0,0261 0,0184 1,9261 0,7189 0,6559 f15 0,0769 0,0511 0,0205 1,8439 4,7573 2,4456 f 16 0,0543 0,0389 0,0223 1,9290 1,5147 1,4182

As the first PCR didn't show the length of all biobricks, the samples failed last time were sent to run another c-PCR. The result was showed in Fig.1

However, only sample F11 shows the band with right size on the gel pic. Samples, F1, F3, F4, F5, F9, F10, F11, F12, F13, F14 and F17 were inoculated again to get enough plasmid for further use. 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, except for F16.

The concentration of plasmid were measured for all the samples and values were showed in Table 2

Plasmid of F1, F2, F4, F9,F10,F12, F17,ccdb C3 and ccdb K3 were digested with proper enzymes bought from Sigma-Aldrich. The biobricks were ligated in pairs according to the plan by Quick Ligase kit from Fermentas. 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.

Construction of G1- G7

5 colonies were picked from each of those plates and sent to perform c-PCR. According to the band size on gel picture, 1 or 2 colonies of each set were selected to inoculate.