Team:Warsaw/Calendar-Main/30 July 2008

Purification of proteins: Z-alpha and Z-omega Piotr, Emilia, Weronika pET15b+Z_alpha and pET15b+Z_omega in Rosetta strain from overnight culture inoculated in fresh LB and cultured until OD=0,5. Cultures induced with different concentrations of IPTG in 22&deg;C and 37&deg;C. Samples were collected twice: after 3h and next day (samples were centrifuged and frozen).

Cloning of omega_&Delta;A DNA fragment to pACYC177+OmpA_alpha Michał K.  Separate transformant colonies (tranformation of ligation of <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_alpha and omega_&Delta;A</a> from previous day) inoculated to liquid LB with kanamycin.

Cloning of truncated fragment of protein A (&Delta;A) Michał K. Optimization of <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> to obtain truncated fragment of protein A DNA. Primers: <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI ">AL+SacI</a> <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> Template DNA: pDRIVE-TapTag Elongation time: 30s

- Optimization of annealing temperature (gradient from 55&deg;C to 75&deg;C). <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/30_July_2008#fig1">Fig. 1</a>.

- Optimization of number of cycles(15, 20, 25, 30, 35). <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/30_July_2008#fig2">Fig. 2</a>.</li>

<a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> to obtain truncated A protein DNA fragment. Primers: <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI ">AL+SacI</a> <a href="http://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> Template DNA: pDRIVE-TapTag Elongation time: 30s

Annealing temperature: 60&deg;C

20 cycles </li>

Gel electrophoresis and <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of 250 bp band. <a href="http://2008.igem.org/Team:Warsaw/Calendar-Main/30_July_2008#fig3">Fig. 3</a>. </li>

<a href=http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of isolated PCR product, <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a> and <a href=http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega>pACYC177+OmpA_omega</a> with NotI and SacI (BamHI buffer). pACYC177 vectors were also <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylated</a>. </li> <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of digest reaction. </li> Gel electrophoresis for estimation of DNA concentration. </li> Overnight <a href="http://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a>: pACYC177+OmpA_alpha + &Delta;A and pACYC177+OmpA_omega + &Delta;A.</li></ol>

<img src="http://2008.igem.org/wiki/images/3/32/Nazwa_pliku.jpg" width=300/></a> Fig. 1. Gradient PCR to obtain truncated protein A (temperatures: 55-75&deg;C) 1. Marker 2. 50&deg;C 3. 55&deg;C 4. 60&deg;C 5. 65&deg;C 6. 70&deg;C 7. 75&deg;C

<img src="http://2008.igem.org/wiki/images/7/75/Plik.jpg" width=300/></a> Fig. 2. PCR to obtain truncated protein A (various number of cycles) 1. Marker 2. 15 cycles 3. 20 cycles 4. 25 cycles 5. 30 cycles 6. 35 cycles

<img src="http://2008.igem.org/wiki/images/c/c4/Go_26_08_2008.jpg" width=300/></a> Fig. 3. PCR amplified truncated protein A 1. Marker 2. PCR product (deltaA), temperature of annealing = 60&deg;C, 20 cycles