Team:Warsaw/Calendar-Main/14 August 2009

Cloning Bax into pSB1A3 plasmid Justyna Task 1:  Gel-out Bax PCR product Methods: Bax PCR products were isolated from agarose gel using A&A Gel-Out kit. Task 2:  Digestion and gel-out of pSB1A3 plasmid  Methods:  Digestion of isolated plasmids by XbaI and SpeI Reaction mixture composition: 10.0 &mu;l purified plasmid DNA product, 0.5 &mu;l XbaI (Fermentas),0.5 &mu;l SpeI (Fermentas), 2 &mu;l Buffer Orange (Fermentas), 7.0 &mu;l MQ water</li> The digestion was performed overnight and it was subsequently inactivated via heating in 80&deg;C for 20 minutes.</li> In the next step reaction mixtures were loaded into the agarose gel to analize restriction pattern of the plasmids and isolated using A&A Gel-Out kit</li></ul> Task 3:  Ligation of Bax and pSB1A3</li></ul> Methods:  Ligation mix:</li> 1.0 &mu;l - T4 ligase 2.5 &mu;l - Tango buffer (Fermentas, 10x) 2.0 &mu;l - dNTPs (EURx, 5&mu;lM) 5.0 &mu;l - digested pSB1A3 (~100 ng) 14.5 &mu;l - Bax (~ 105 ng) in 25.0 &mu;ll Ligation time - overnight</li></ul> Task 4:  Insertion of Bax-pSB1A3 plasmid into bacteria</li></ul> Methods:  Previously prepared chemicompetent bacteria were used.</li>

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Assembly of endosomal detection operon Marcin Task 1:  Isolate the plasmid prepared in <a href="http://2009.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/13_August_2009">13.08.09</a></ul></li> Methods:  Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described <a href="http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf">here</a></li></ul> Task 2: <li>Restriction digest of following sequences from the <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid: <ul> <li>p53 CDS</li> <li><a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> +<a href="http://partsregistry.org/Part:BBa_C0040"> BBa_C0040</a> </li> <li><a href="http://partsregistry.org/Part:BBa_R0080"> BBa_R0080</a> <li><a href="http://partsregistry.org/Part:BBa_E0032"> BBa_E0032</a> </li></ul> </ul></li> </li> </ul> Methods: <ul> <li> Reaction mixture composition:</li> 20 &mu;l purified plasmid DNA product 1 &mu;l XbaI (Fermentas) or 0,5 &mu;l SpeI (Fermentas) in the case of R0080 1 &mu;l PstI (Fermentas) 5 &mu;l Buffer Tango (Fermentas) 24 &mu;l MQ water </ul> Task 3: <ul><li>Gel-out of digest sequences described in Task 1</ul></li> Methods: <ul> <li>Fragments of agarose gel were carefully cut out and subsequently frozen in -20&deg;C></li></ul> Results <img src="http://2009.igem.org/wiki/images/9/9a/P53_R0080_E0022_C0040_B0032_digest_14_08_09.png" width="40%" height="40%"> <font face="Times New Roman" size="3"> Verification of the digestions Comment: Restriction digests of samples containing <a href="http://partsregistry.org/Part:BBa_E0022"> BBa_E0022</a> and <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> +<a href="http://partsregistry.org/Part:BBa_C0040"> BBa_C0040</a> </li> were unsuccessful, probably the probes were contaminared. Because of this fact I decided to repeat the digest. Task 4: <ul><li>Another digest of E0022 and C0040+B0032</ul></li> Methods: <ul> <li> Reaction mixture composition:</li> 20 &mu;l purified plasmid DNA product 1 &mu;l XbaI (Fermentas) 1 &mu;l PstI (Fermentas) 5 &mu;l Buffer Tango (Fermentas) 24 &mu;l MQ water </ul> Task 5: <ul><li>Transformation of chemocompetent E. coli strain DH5&alpha</ul></li> Constructs to transform: </ul><ul> <li>cro CDS + <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> </li> <li><a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> + <a href="http://partsregistry.org/Part:BBa_E0022"> BBa_E0022</a> </li></ul> Methods: <ul> <li>Ligation prepared in 08.08.09 was stopped via thermal inactivation in 80&deg;C for 20 minutes</li> <li>Detailed protocol of transformation is described <a href="http://2009.igem.org/Team:Warsaw/Calendar-Main/5_July_2009">here</a>.</li> </ul> Task 6: <ul><li>Prepare chemocompetent TOP10 bacteria strain (I worked with Kuba)</ul></li> Methods: <ul> <li>Detailed protocol is described <a href="http://2009.igem.org/Team:Warsaw/Calendar-Main/22_July_2009">here</a></li>