Team:Warsaw/Calendar-Main/13 June 2008

Blue/white and rifampicin test #1 Piotr

AID in transcription fusion works like AID, but induce slower growth than AID; AID in translation fusion do not induce, or weakly induce rifampicin resistance mutation

Preparation of constructs: OmpA_alpha and OmpA_omega

Michał K. PCR on pB30D plasmid using

OmpaL_N and OmpaP_link

primers (elongation length 45s). Fig. 1 and Fig. 2.  PCR</a> on pUC19</a> plasmid using

AlphaL_link</a> and AlphaP_XB</a> primers (elongation length 45s). Fig. 2</a> and Fig. 3</a>.</li> PCR</a> on pUC19</a> plasmid using OmegaL_link</a> and OmegaP_EPB</a> primers (elongation length 30s). Fig. 3</a> and Fig. 4</a>.</li> Each reaction was carried out with 15 and 20 cycles and in temperature gradient from 55 to 75 degrees.

<img src="http://2008.igem.org/wiki/images/2/2b/PCR_Ompa_Alpha_Omega_WAW1.jpg" width=400/> </a> <img src="http://2008.igem.org/wiki/images/8/82/PCR_Ompa_Alpha_Omega_WAW2.jpg" width=400/> </a> <img src="http://2008.igem.org/wiki/images/7/76/PCR_Ompa_Alpha_Omega_WAW3.jpg" width=400/> </a> <img src="http://2008.igem.org/wiki/images/6/68/PCR_Ompa_Alpha_Omega_WAW4.jpg" width=200/></a> Gradient PCR products: 1, 14 and 27-DNA ladder; 2 to 8 - gradient PCR for Alpha carried out with 15 cycles; 9 to 16 - gradient PCR for Alpha carried out with 20 cycles; 17 to 23 - gradient PCR for Omega carried out with 15 cycles; 24 to 31 - gradient PCR for Omega carried out with 20 cycles; 32 to 38 - gradient PCR for OmpA carried out with 15 cycles; 39 to 46 - gradient PCR for OmpA carried out with 20 cycles. Product od each variant of PCR is in 7 lanes: 1st is the lower border annealing temperature (55 degrees), 6th is the higher annealing temperature (75 degrees) and 7th is negative control without template DNA.