IGEM:UNAM-Genomics Mexico/2009/Notebook/H2 Pae/2011/07/12

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Isolation of the Standard pBRRMCS-5 from sp. BL21 From the yesterday's transformation, 2 colonies grew in the first reaction and 10 colonies grew in the second one. Paulina take the two first and two from the second reaction and she put them to grow in liquid medium, 37°C in agitation. Six and a half hours later I did the plasmid isolation following this protocol. The results are shown in an electrophoresis gel (80V x 100 min): Lanes: Ladder 10 Kb Isolation 1 Isolation 2 Isolation 3 Isolation 4 This shows that the plasmid isolation is a little bit contaminated, but it wont affect following procedures. Double digestion In order to see if our PCR was correctly inserted into pBBRMCS-5, I did a double digestion with the same enzymes used to generate sticky ends between the plasmid and the PCR, these enzymes are apaI and sacI. Digestion's proportions: Reagent microL H20 4 DNA 2 BSA 100x 1 Buffer 4 10x 1 apaI 1 sacI 1 Total 10 The reaction was left overnigth, 25 °C