Team:Panama/9 August 2010

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Revision as of 23:40, 14 October 2010

August 9

High resolution agarose gel (for small fragments). For this we used two lanes (1 and 5) for the molecular weight. We used the Lamda DNA/Hind III marker from Biotools. We decided to run duplicates of the samples, so we have the Promoter and RBS on lanes 2 & 3; lane 4 is empty and once again, the Promoter and RBS are in lanes 6 & 7.

Ethydium Bromide staining:

The stock concentration was at 10mg/ml.

The concentration for gel staining was 0.5ug/ml.

We used 15ul of Ethydium Bromide in 300ml of dH2O. We stained it covered for 30 minutes. Afterwards, we washed it in water for 10 minutes each time.

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Digestion Repetitions:

1. Promoter (using iGEM restriction enzymes)

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Buffer2 = 5ul

BSA = 0.5ul

EcoR1 = 1ul

Spe1 = 1ul

DNA = 17.98 ul

dH2O = 24.52

Final volume = 50ul

2. Promoter (using INDICASAT restriction enzymes)

Buffer2 = 5ul

BSA = 0.5ul

EcoR1 = 1ul

Spe1 = 1ul

DNA = 17.98 ul

dH2O = 24.52ul

Final volume = 50ul

3. RBS

Buffer2 = 5ul

BSA = 0.5ul

Xba = 1ul

Pst1 = 1ul

DNA = 11.90ul

dH2O = 30.6ul

Final volume = 50ul

The reason behind testing different batches of the same restriction enzymes for the promoter was to find out the reason why we didn't see anything when we ran electroforesis gels. We had thought that maybe the restriction enzymes were not doing their job, so we tried digesting the parts with enzymes that INDICASAT provided.

PsB1C3 Plasmid transformation (Stock):

• LB plates with Cloranfenicol at a concentration of 33ug/ml.

• LB plates with Tetracycline at a concentration of 15ug/ml.