Team:Warsaw/Calendar-Main/20 July 2009

Insertion of the pho gene into the pKSII+ plasmid Kama  Plasmid digest mix was prepared as follows: 3&mu;l Tango buffer (Fermentas) 4&mu;l pKSII plasmid 1&mu;l XbaI enzyme 1&mu;l SmaI enzyme The solution was topped up with H2O to the final volume of 30 &mu;l. Pho gene digest mix was prepared as follows: 3&mu;l Tango buffer (Fermentas) 16&mu;l purified gene 1&mu;l XbaI enzyme The solution was topped up with H2O to the final volume of 30 &mu;l. The digest was kept for 2h at 30&deg;C, after that 2h at 37&deg;C and then the enzymes were inactivated for 20min. at 70&deg;C.  The ligation mix was prepared as follows: 2&mu;l plasmid 8&mu;l gene 3&mu;l ligation buffer 1 &mu;l T4 DNA ligase (Fermentas) 0,5&mu;l PEG The solution was topped up with H2O to the final volume of 30 &mu;l. As a control: 2&mu;l plasmid 3&mu;l ligation buffer 1 &mu;l T4 DNA ligase (Fermentas) 0,5&mu;l PEG The solution was topped up with H2O to the final volume of 30 &mu;l. The ligation was carried out in 18&deg;C overnight (~15h)

Construction of K177012 operon1_part2

Ania

Tasks:


 * Set up a liquid culture from transformants containing BBa_B0032 - RBS.3 and BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid in order to amplify the plasmid and extract it from the bacteria.

Comment:
 * Accidentally I used the whole DNA samples extracted on Thursday to perform the digestion. I Added the digestion mix to my samples, hence the wrong buffer concentration and improper digestion on Friday. Thinking saves time and money. But there is another reason to justify the repeated plasmid extraction. Now I know that my transformants after all have the correct plasmid so I can do the alkaline lysis on very few samples and use the AA kit to get greater DNA concentration. After all this is my insert for the next ligation, so we need a lot. I know that no one will ever read the notebook up to this point.

Testing different E. coli strains regarding lacI and AraC repressors
Franek

Tasks:


 * Seting liquid cultures of Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a competent cells with BBa_K177024 and BBa_K177025

Methods:


 * 7 test tubes with 5ml LB were inoculated with Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a. The cultures were left for overnight incubation at 37°C.


 * 14 test tubes with 5ml LB and ampicillin were inoculated with Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a, containing either BBa_K177024 or BBa_K177025 on pSB1A2 plasmid. The cultures were left for overnight incubation at 37°C.


 * 7 test tubes with 5ml LB, ampicillin and IPTG were inoculated with Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a, containing BBa_K177024 on pSB1A2 plasmid. The cultures were left for overnight incubation at 37°C.


 * 7 test tubes with 5ml LB, ampicillin and 0,2% arabinose were inoculated with Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a, containing BBa_K177025 on pSB1A2 plasmid. The cultures were left for overnight incubation at 37°C.

Results:


 * Will be determined next day by comparing GFP level in diffrent cultures

Cloning of the mgtc promoter into the pKSII+ plasmid Kamil

Tasks:  Plasmid assembly 

Methods:  Plasmid digest mix was prepared as follows: 2&mu;l Tango buffer (Fermentas) 3&mu;l pKSII+ plasmid 2&mu;l XbaI enzyme (Fermentas) 2&mu;l SmaI enzyme (Fermentas) The solution was topped up with H2O to the final volume of 20 ul.</li> mgtc promoter digest mix was prepared as follows: 2&mu;l Tango buffer (Fermentas) 10&mu;l purified gene 2&mu;l XbaI enzyme The solution was topped up with H2O to the final volume of 20 ul.</li> The digest was kept for 6h (to compensate for the 50% loss in activity of the SmaI enzyme) at 37&deg;C, and then the enzyme was inactivated for 15min. at 80&deg;C.</li>  The ligation mix was prepared as follows: 10&mu;l of both inactivated digests were mixed together and topped with 2&mu;l of 30% PEG 2,2&mu;l ligation buffer (containing ATP) 2&mu;l of T4 ligase (Fermentas). The ligation was carried out in 18&deg;C overnight (~18h) and then inactivated for 10min. at 65&deg;C.</li> </ul>

Results: <img src=http://2009.igem.org/wiki/images/d/d7/2009.07.20.jpg /> From left: <ol> GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> Ligation</li> Undigested plasmid</ol>

Conclusions:  Despite the low morale we decided we could transform with that.</li> </ul>

Assembly of endosomal detection operon Marcin

Task 1:  Inactivation of ligation</li></ul> Ligation mixtures prepared 19.07.09 were inactivation via heating in 80&deg;C for 20 minutes. Comment: Due to some doubts about quality of the chemocompetent bacteria I decided to freeze the ligation mixtures until the situation would become more clear.

Cloning of p53 coding sequence Marcin Task 1:  Prepare PCR reaction to amplified p53 coding sequence.</li> </ul> Methods:  DNA template dilution:</li> </ul> 1 &mi;l of plasmid solution (isolated in 11.07.09) was taken and diluted with 99 mi&;l of MQ water  PCR mixture composition:</li> <ol> <li>proper mixture 1: 0.25 &mu;l primer 1 (50 nM; Oligo.pl) 0.25 &mu;l primer 2 (50 nM; Oligo.pl) 1.5 &mu;l dNTPs (20 &mu;M ;Fermentas) 0.5 &mu;l Pfu turbo polymerase (KNGiE) 2.5 &mu;l Pfu Turbo Buffer (Fermentas) 2.5 &mu;l MgSO4 (20 &mu;M; Fermentas) 1 &mu;l DNA template 16.5 &mu;l MQ water </li> <li>proprer mixture 2: 0.25 &mu;l primer 1 (50 nM; Oligo.pl) 0.25 &mu;l primer 2 (50 nM; Oligo.pl) 1.5 &mu;l dNTPs (20 &mu;M ;Fermentas) 0.5 &mu;l Pfu turbo polymerase (KNGiE) 2.5 &mu;l Pfu Turbo Buffer (Fermentas) 2.5 &mu;l MgSO4 (20 &mu;M; Fermentas) 2 &mu;l DNA template 15.5 &mu;l MQ water</li> <li>Negative control: the same as proper mixture 1, the only distinction is lack of the DNA template.</li> </ol></ul> <ul> <li>Program:</li> p53 (detailed destription is <a href="http://2009.igem.org/Team:Warsaw/Calendar-Main/9_July_2009">here</a>) Results: No product obtained Comment: Previous PCR reaction were prepared using another thermocycler with distinct intristic parameter than used in this case. Perhaps, if the reactions were prepared in that device the results would be better.