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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
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.
Taís
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