Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2016/03/01

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03/01/2016

Transforming a Negative Sender Control Strain

Jiaqi and I transformed an MSV negative control sender. Two plates -- One with the MSV control and the other the water-only control -- were incubated at 37°C overnight.


Digesting pSB1A3

The pSB1A3 plasmid (http://parts.igem.org/Part:pSB1A3) was digested with XbaI and SpeI restriction enzymes to remove mCherry from the backbone. We used gel purification to make sure that the insert did not re-ligate with the backbone. The following reagents were used in the amounts specified in the table below to prepare the plasmid for digestion:

Table 1 - pSB1A3 Digestion Reaction Mixture
Reagent Volume (µL)
DNA (62 ng/µL) 15
XbaI 1
SpeI 1
FD Buffer 3
Water 10
Total 30

We used a larger amount of DNA than usual to account for the fact that gel extraction usually results in low yields. The restriction enzymes were not heat inactivated after this protocol, as the digestion was immediately followed by gel purification. The gel with the negative control receiver was run for an hour in total. The plasmid was broken up into 3 bands, with the band around 3000 bp's representing the uncut backbone, the one around 2000 bp's representing the cut backbone, and the one around 1000 bp's representing the excised mCherry component of the plasmid, as shown in the picture below:

Figure 1 - pSB1A3 Gel Purification
:pSB1A3 Gel Purification

The gel purification was followed by gel extraction, determination of DNA concentration, ligation, and transformation on Wednesday, 2 March 2016.


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