*<p style=”text-align:justify;”> We made the digestion of BBa K112806 with EcoRI and SpeI, BBa B0015 with EcoRI and XbaI and BBa I746911 with SpeI and Pst. </p>
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*<p style=”text-align:justify;”> We purified the digestion and made the ligation of BBa K112806 in the BBa B0015 plasmid according to the [https://2009.igem.org/Team:UNICAMP-Brazil/Protocols/T4_DNA_Ligase Protocol 11] </p>
Ligation of finOP and Cre-Recombinase on pGEM vector
In order to start our pGEM strategy on assembling biobricks, today we ligated PCR's products of finO, finP and Cre-Recombinase into pGEM Vector. There is no need for any digestion before this procedure, since pGEM has an T stick ends capable of pairing with the A stick ends left by Taq Polymerase on the PCR products.
Transformation of finOP's and Cre-Recombinase's ligations
After performing the required ligations, we then transformed them into electrocompetent E. coli bacteria, strain DH10B, according to Protocol 3.
We plated the trasformed cells in LB-AMP media, which already contained X-gal substrate.
Plates were incubated at 37ºC for an O/N period.
Marcelo and Victor
Kamikaze’s parts digestion and ligation
We made the digestion of BBa K112806 with EcoRI and SpeI, BBa B0015 with EcoRI and XbaI and BBa I746911 with SpeI and Pst.
We purified the digestion and made the ligation of BBa K112806 in the BBa B0015 plasmid according to the Protocol 11
Marcos
YeastGuard
New biobricks - New strategy (pGEM)
The ligation reactions didn´t work. So today we decided not to digest the fragment and the pGEM vector with SpeI, but to ligate it directly, considering that we can confirm the correct insertion by PCR.
We did new ligation reactions with our four parts (pJEN1, pDLD, Lysozyme and orfJEN1) in pGEM.
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