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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
f16	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

Analysis

Collection time 2010-08-05 14:34:11

   Sample        A[260]     A[280]    Bg[320]     Ratio     Protein   Nucleic Acid 

                                                             µg/ml       µg/ml     

____________________________________________________________________________________

f1 0,0183 0,0101 -0,0027 1,6428 3,9441 86,2531

f2 0,0477 0,0334 0,0160 1,8282 2,9083 137,1389

f3 0,0255 0,0132 -0,0021 1,8045 2,8374 118,5919

f4 0,0518 0,0398 0,0216 1,6573 5,4159 124,4569

f5 0,0108 0,0033 -0,0041 2,0017 0,2684 66,7742

f6 0,0137 0,0079 0,0006 1,7979 1,3839 56,0149

f7 0,0072 0,0010 -0,0071 1,7638 1,7529 60,7466

f8 -0,0035 -0,0080 -0,0151 1,6180 2,3362 47,0326

f9 0,0104 0,0035 -0,0054 1,7835 1,7833 67,4654

f10 0,0074 -0,0004 -0,0076 2,0846 -0,1927 68,6519

f11 0,0184 0,0088 -0,0063 1,6384 4,6986 101,2263

f12 0,0008 -0,0044 -0,0115 1,7349 1,6832 51,6802

f13 0,0157 0,0066 -0,0074 1,6499 4,2257 94,6587

f14 0,0033 -0,0031 -0,0100 1,9215 0,6691 58,6173

f15 0,0403 0,0345 0,0294 2,1530 -0,3966 50,2377

16 0,0182 0,0075 -0,0052 1,8335 2,0872 101,2592

f17 0,0082 0,0036 -0,0020 1,8332 0,9140 44,2838

Concentration before dilution for dection

ng/µL

f1 52

f2 82

f3 71

f4 75

f5 40

f6 34

f7 36

f8 28

f9 40

f10 41

f11 61

f12 31

f13 57

f14 35

f15 30

f16 61

F17 27

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.