08/07/2014

What’s up y’all? =)

Today a plasmidial extraction was made by using the inocullum kit of JM110 transformed with Bioremediation Biobrick; DH5α transformed with Bioremediation Biobrick; DH5α transformed with the synthesis Essential Biobrick; and JM110 with pUC72

Eletrophoretical profile of the extraction:

JM110 Bioremediation Biobrick 1

1. JM110 Bioremediation Biobrick 2

2. DH5α Bioremediation Biobrick 1

3. DH5α Bioremediation Biobrick 2

4. DH5α Essential Biobrick 1

5. DH5α Essential Biobrick 2

6. pUC72 ofJM110

7. pUC72 of JM110

After the extraction, we digested the samples: 2 ( Bioremediation Biobrick ) with EcoRI and XbaI to linearize the plasmid; 5 (Essential Biobrick) with EcoRI and SpeI to release the insert we needed. We used the following system:

A digestão durou 2 horas em 37°C. Quantidade aplicada no gel foi de 10ul para cada amostra e 40 ul foi armazenado em -20°C para posterior purificação.

1. Biobrick para Biorremediação de JM110 (controle/não digerido).

2. Biobrick para Biorremediação de JM110 digerido com EcoRI e XbaI

3. Biobrick Essencial de DH5α (controle/não digerido).

4. Biobrick Essencial de DH5α digerido com EcoRI e SpeI

It digested!!! We can observe that sample 2 is linearized and sample 4 shows the fragment we wanted to isolate! However, when analyzing electrophoretic profile of sample 2, we conclude that there was chromosomal DNA together with plasmid extraction, contaminating our digestion. Because of this, we again inoculated the transformants to remake plasmid extraction and enzymatic digestion.