Colonies had grown on plates as followed:

• LB agar: more than 1000 colonies for the two biobricks
• LB agar kanamycin: 10 colonies for BBa_E0240 and >40 colonies for BBa_I20260
• LB agar chloramphenicol: >50 colonies for BBa_E0240 and 3 colonies for BBa_I20260

Plate storage at 4°C.

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Nothing had grown during the night on both plates. Taking into consideration different hypothesis, the transformation was repeated with a change on step 6 and 7. At step 6, only 1 ml of LB medium was added. To plate, 200 µl of BBa_E0240 and BBa_I20260 were plated on 3 plates: LB agar, LB agar kanamycin and LB agar chloramphenicol.

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To amplify BBa_I20260 and BBa_0240, a PCR was perform with the mix described Table 1. The program IGEM Q5 PCR was applied, see Table 2. PCR products were cleaned with the GeneJET purification kit (Thermo Scientific)followed by DNA quantification with the NanoDrop 2000 (Thermo Scientific).

Preparation of a 1% agarose gel: 0.52 g of Top Vision agarose (Thermo Scientific) + 50 ml of TAE 1X. Microwave 30s by 30s until agarose total dissolution Gel was cooling down until to be lukewarm, one BET drop was added. Gel was loaded with 20 µl of BBa_E0240 PCR product and BBa_I20260 purified PCR product added with 4 µl of loading dye, at 10µl of the mix per well. Gel running 45 minutes at 100 mV in TAE 1X buffer.

Figure 4: 1% agarose gel of PCR products from 08.14.2014 after PCR clean up. Lane 1 and 5: Purple 2-Log ladder NEB, Lane 2, 3 and 4: BBa_E0240 purified PCR product, Lane 6,7 and 8: BBa_I20260 purified PCR product

3 pieces of gel were sampled in four tubes to perform a DNA purification from gel. The DNA purification was made with the GeneJET purification kit (Thermo Scientific.
A verification electrophoresis was performed on a 1% agarose gel.

Figure 5: 1% agarose gel of purified fragments from gel. Lane 1: Purple 2-Log ladder NEB, Lane 2: BBa_E0240 1200 bp purified fragment and Lane 3: BBa_I20260 1200 bp purified fragment

The major part of the DNA was lost during the purification because the amount of was to weak to be purified from gel. So we decide do transform ''E. coli'' to isolate a colony containing the desired plasmid for BBa_E0240 and BBa_I20260.

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Final Section

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