08/27/2014

Hello, dear reader! Today we made plasmid DNA extraction from the amplified colonies (8 and 11) in PSB1C3 our BB_Essential + BB_Bioremediation.

Electrophoretic plasmid DNA extraction’s profile:

1 – 3: BB_Essential + BB_Bioremediation in PSB1C3 extracted from DH5a colony 8.

3 – 6: BB_Essential + BB_Bioremediation in PSB1C3 extracted from DH5a colony 11.

To confirm that we’re really with all our bio bricks BB_Essential + BB_Bioremediation, biobrick BB_Essential and biobrick BB_Bioremediation (done previously) in PSB1C3, we will cut with EcoRI and PstI to analyze the fragment’s size that will be released and the vector. We kept the same digestion systems of previous days for 1:30 hours at 37°C.

Electrophoretic profile of enzymatic digestion with EcoRI and PstI:

1: PSB1C3 with BB_Bioremediation not digested

2: PSB1C3 with BB_Bioremediation digested with EcoRI e PstI colônia 2

3: PSB1C3 with BB_Bioremediation digested with EcoRI e PstI colônia 3

4: PSB1C3 with BB_Essential not digested

5: PSB1C3 with BB_Essential digested with EcoRI e PstI colônia 2

6: PSB1C3 with BB_Essential digested with EcoRI e PstI colônia 4

7: PSB1C3 with BB_Essential + BB_Bioremediation not digested

8: PSB1C3 with BB_Essential + BB_Bioremediation digested with EcoRI e PstI colônia 8 A.

9: PSB1C3 with BB_Essential + BB_Bioremediation digested with EcoRI e PstI colônia 8 B.

9 – 11: Duplicate of 7, 8 and 9 with colonies 11 A and B.

Uhuuu!! Super digestion!! As we could see, the samples 4-11 show the expected profile, meaning the presence of it respective fragments and vectors. What intrigued us were the samples 1, 2 and 3. But we’ve passed through this before. What happened is that the fragment has about the same size as the vector, making it difficult to separate them using agarose gel.

To solve this problin, we’re going to digest only using EcoRI to make it linear and with EcoRI + PstI to show the fragment and the vector. If the one digested with EcoRI is the sum of both fragment + vector, it is right! We bet it is! What do you think?!?! We’ll have the answer tomorrow.