Team:UNICAMP-Brazil/Notebooks/October 3

ColiGuard

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.

• We followed Protocol 11, without modifications.

• Ligation lasted 1 hour.

Marcelo and Victor

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 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.

Taís