08/07/2014

What’s up y’all? =)

Today a plasmidial extraction was made by using the inoculum 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 made digestion of 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:

The digestion took 2 hours at 37°C. The amount applied on gel was 10ul of each sample and 40ul was stocked at -20°C for posterior purification.

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 was 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 concluded that there was chromosomal DNA together with plasmid extraction, contaminating our digestion. Because of this, we again inoculated the transforming cells to remake plasmid extraction and enzymatic digestion.