{ "date" : "2013-06-18", "author" : "gabriele-emil", "title" : "SAMsynthetase: episode 2
Attack of the Insert", "content" : "This morning we added 1µl of SAP to the pSB1C3 overnight digestion and 1µl of DpN1 to the SAMsynthetase overnight digestion, then both were incubated at 37°C for 1.5 hours.

During these 1.5 hours, we performed the miniprep (protocol) and quantification of the circular pSB1C3 inocula of yesterday.

Circular pSB1C3 quantification results
G1184.1 ng/µl
G2187.7 ng/µl
G3165.7 ng/µl
E1117.3 ng/µl
E2105.7 ng/µl
E399.0 ng/µl

After the incubation, we purified the digestion mixes using the Wizard® SV Gel and PCR Clean-Up System and then quantified.
Quantification results
linear pSB1C330.0 ng/µl
SAMsynthetase G140.4 ng/µl
SAMsynthetase E132.8 ng/µl

SAMsynthetase E1 was stocked at -20°C.

Then, we performed the ligation of pSB1C3 and SAMsynthetase exploiting the usual protocol.
Ligation mix
We incubated the ligation mixes for 2 hours at room temperature.

Also, we extracted R0010 promoter (Plac) from the registry (2013 distribution kit, plate 3, well 3H). Unfortunately, we also extracted a part from the same plate and well of the 2012 distribution kit (BBa_K115032) because we mistook the kits.

Finally, we transformed NEB10β competent cells with the usual protocol, we used the following quantities of DNA: 10µl of each ligation product except for the 1:4 ligation, of which we used 15µl, and 1µl of the extracted R0010. Then, we plated on CM plates.", "tags" : "SAMsynthetase" }