Team:Warsaw/Calendar-Main/13 August 2009

Amplyfing of Bax sequence Justyna Methods  PCR reaction mix: per 50μl: 5.0 μl - 10 x buffer (20mM MgSO4) 3.5 μl - dNTP mix (5mM) 3.0 μl - primer BaxF (0.5 μM) 3.0 μl - primer BaxR2 (0.5 μM) 1.0 μl - template DNA 32.0μl - mQ water 2.5 μl - Yellow PfuPlus DNA polymerase (1U/μl) Template DNA in 3 dilutions - 101 (pure plasmid DNA), 10-1 and 10-2. buffer, dNTP mix and polymerase from EURx. DNA template - human Bax with changed codons to be more efficient in yeast.  Thermal cycling conditions for PCR (~7-8 kb): 94°C, 1 min 0s

94°C, 0 min 15s 69°C-74°C, 0 min 30s 68°C, 1 min 0s cycles 1-10

94°C, 0 min 15s 69°C-74°C, 0 min 30s 68°C, 1 min 20s cycles 11-25

68°C, 7 min 0s 4°C, indefinite Results: Best reaction for 101 and 10-1 (more specific), in 69.1°C and 69.8°C.  

Cloning of the mgtc promoter into the pSB1A3 plasmid Kamil

Tasks:  Reamplification of the mgtc promoter 

Methods:  The PCR mix was prepared as follows: 5&mu;l buffer B (EURx) 2&mu;l 5mM dNTPs (EURx) 5&mu;l forward starter 5&mu;l reverse starter 2&mu;l OptiTaq polymerase (EURx) 2&mu;l matrix 29 &mu;l H2O </li></ul> PCR programme: 4min 95&deg;C (30s 95&deg;C, 35s 58&deg;C, 40s 72&deg;C)x28 10min 72&deg;C ~ 4&deg;C </li></ul>  The results were visualised with gel electrophoresis on 1% agarose gel.</li> The appropriate bands were extracted using Gel-Out kit (A&A Biotechnology) according to the producers protocol. </ul>

Results:  Gel Electrophoresis:</li> </ul> <img src=http://2009.igem.org/wiki/images/3/30/2009.07.27.jpg /> From left: <ol> GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> 50&mu;l of PCR mix</li> 50&mu;l of PCR mix</li> Negative control</li> </ol>

Conclusions:  Reamplification was successful.</li> </ul>

Assembly of endosomal detection operon Marcin

Task 1: <li>Gel-out of samples which was cut out from the gel in <a href="http://2009.igem.org/Team:Warsaw/Calendar-Main/12_August_2009">12.08.09</a></li></ul> Procedure: <ul> <li>Fragments of agarose gel were carefully cut out and in the next step DNA was extracted from the gel using the A&A gel-out kit. Detailed procedure is described <a href="http://aabiot.com/products/dna_purification/dna_fragments/gel_out/protocol_gel_out.pdf">here</a></li></ul> Results: <img src="http://2009.igem.org/wiki/images/0/01/Gel_outs_13_08_09.png" width="40%" height="40%"> <font face="Times New Roman" size="3"> Evaluation of gel-outs yield Comment: The concentration of DNA is sample containing <a href="http://partsregistry.org/Part:BBa_R0080"> BBa_R0080</a> was surprisingly low (sample 1). Because of this I decided to repeat the digest and try to use another gel-out kit. Task 2: <ul><li>Restriction digest of <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> </li></ul> Methods: <ul> <li>Digest of BBa_R0080 using SpeI and PstI</li> <li> Reaction mixture composition:</li> 20 &mu;l purified plasmid DNA product 0.5 &mu;l SpeI (Fermentas) 1 &mu;l PstI (Fermentas) 5 &mu;l Buffer Tango (Fermentas) 24 &mu;l MQ water </ul> Task 3: <ul><li>Cloning the sequences: cro CDS and <a href="http://partsregistry.org/Part:BBa_E0022"> BBa_E0022</a> with <a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> vector</ul></li> Methods: <ul> <li> Ligation mixtures composition:</li> 15 &mu;l digested cro (or E0022) 8 &mu;l digested vector 3 &mu;l Tango buffer(Fermentas) 3 &mu;l dNTPs mixture (EurX, concentration 5 mM) 1 &mu;l ligase T4 (Fermentas) <li>Duration of ligation was about 18 hours; reaction was conducted in 19 &deg;c (approximately).</li> <li>In the next step ligated samples were thermally inactivated via heating in 80 &deg;c for 20 minutes</li> </ul> Task 4: <ul><li>Prepare the bacterial cultures for isolation of on <a href="http://partsregistry.org/Part:pSB1A3"> pSB1A3</a> plasmid containing:</li> <ul> <li>p53</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_B0032"> BBa_B0032</a> +<a href="http://partsregistry.org/Part:BBa_C0051"> BBa_C0051</a> </li> <li><a href="http://partsregistry.org/Part:BBa_B0032"> BBa_B0032</a> +<a href="http://partsregistry.org/Part:BBa_E0032"> BBa_E0032</a> </li></ul>