Team:Warsaw/Calendar-Main/8 July 2009

Gradient PCR Pho Kama

Tasks:  Amplification of phoP/phoQ 

Methods:  PCR mixture's composition: 1&mu;l pfu buffer (Fermentas) 1&mu;l MgSO4 (Fermentas) 0,5&mu;l primers 0,5&mu;l dNTPs (10 mM) 0,25&mu;l pfu turbo polymerase 0,5&mu;l template DNA from Listeria??? (Salmonella) optionally: 0,75&mu;l DMSO The solution was topped up with H2O to 10&mu;l.    PCR programs: pho 4min 95&deg;C (30s 95&deg;C, 1min 45-55&deg;C, 4min 72&deg;C)x3 (30s 95&deg;C, 1min 55-60&deg;C, 4min 72&deg;C)x28 10min 72&deg;C ~ 7&deg;C   Electrophoretic separation on 1% agarose gel 

Results: <img src="http://2009.igem.org/wiki/images/9/9b/2009_07_09_pho.JPG"/>  Gel (from left)</li> </ul> <ol> control -</li> 2-6 - samples (annealing temperature increases to from the left to the right)</li> M - GeneRuler DNA Ladder Mix #SM0333 (Fermentas) 8-12 - samples with DMSO (annealing temperature increases to from the left to the right) M - GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> </ol> Notes:  many unspecific products were obtained (product of the correct lenght marked with an arrow)</li> </ul>

Quality check (cro PCR product and pKS isolate)

<a name="Kuba"></a> Kuba 

pKS-CRO cut with XbaI and EcoRI</li> </ul>

<img src="http://2009.igem.org/wiki/images/4/4d/PKS-cro(XbaI%2C_EcoRI).JPG" />  Gel (from the left)</li> </ul> <ol> GeneRuler DNA Ladder Mix #SM0333 (Fermentas)</li> uncut plasmid (isolate no.1)</li> cut plasmid (isolate no.1)</li> <li>uncut plasmid (isolate no.2)</li> <li>cut plasmid (isolate no.2)</li> <li>uncut plasmid (isolate no.3)</li> <li>cut plasmid (isolate no.3)</li> </ol> </ol> </ul>

Note: isolate no.1 contains a correctly inserted PCR product

Cloning of p53 coding sequence Marcin

Comment: There were some difficulties with ligation p53 coding sequence into pKS plasmid so I decided to take another sample containing isolate amplified p53 (via PCR reaction) and perform digest of the sample using XbaI Tasks: <ul> <li>Restriction digest of p53 coding sequence obtained from PCR reaction</li> </ul> Methods: <ul> <li>Reaction mixture composition</li> </ul> 22.5 &mu;l PCR product (DNA concentration about 6.5 ng/&mu;ll) 2.5 &mu;l Tango Buffer (Fermentas) 0.5 &mu;l XbaI (Fermentas) <ul> <li>Digest program:</li> </ul> Digest 3h 37 &deg;C 15 min 80 &deg;C ~4 &deg;C <ul> <li>Quantification of the amount of DNA after digest on the agarose gel</li> </ul> Methods: <ul> <li>Electrophoresis of the digested DNA sample:</li> </ul> Conditions: agarose concentration - 1% voltage - 70V time - about 30 minutes After electrophoresis gel was irradiated with UV light and photographed: <img src="http://2009.igem.org/wiki/images/4/4b/P53_08_07_09.jpg" align="center" widht="80%" height="80%"> There is no significant amount of DNA in the samples

Comment:

The sample must have degraded after gel-out procedure. Most probably due to acidic condition in the solution DNA hydrolysed. It is obligatory to do another PCR reaction using the old plasmid sample (I hope the DNA is not degraded!).

Miecznikowa team Aim: debug RFP-terminator ligation that did not work Jarek/Franek/Ania Task1: <ul> <li>Alkaline lysis of the plasmid containing Terminator</li> </ul>

Methods: <ul><li> PlasmidMini set by A&A Biotechnology was used. 2 test tubes with 5ml LB and 5 ul Ampicyline were first  inoculated with the plasmid containing colonies. The cultures were incubated over night at 37C.Alkaline lysis was performed on both cultures. The pellet from 5 ml of bacteria was used. </li> </ul> Results: <ul> <li>DNA extraction quality controll.DNA Concentration was measured using Spectrophotometer NanoDrop ND-1000 </li> </ul> Task2: <ul> <li>We decided to measure the concentration of DNA in our samples for future use e.g. efficient ligation mix. NanoDrop ND-1000 was used.</li> </ul>

Results: Notes - IMPORTANT: <ul> <li>Rfptra and RBSdigested samples were extracted from the gel using gel-out kit by A&ABiotechnology. Presented data shows that after gel extraction there is very little DNA in the sample. Probably there is a ****problem with gel extraction procedure**** - the Gel-out set by A&A Biotechnology might be defective. </li> </ul>

Miecznikowa team - division of labour;) Today the intermediate bricks were designed and we divided the tasks as follows: <ul> <li> Franek: <a href="http://partsregistry.org/Part:BBa_J5528"> BBa_J5528</a>, digestion with (EcoRV, PstI) and insertion into pKS2 (Bluescript) cut with SmaI i PstI (look for white colonies on the X-gal, IPTG, Amp plates) </li> <li> Jarek: construct <a href="http://partsregistry.org/Part:BBa_K177011"> BBa_K177011</a> </li> <li> Michał: construct <a href="http://partsregistry.org/Part:BBa_K177012"> BBa_K177012</a> </li> <li> Marek: construct <a href="http://partsregistry.org/Part:BBa_K177013"> BBa_K177013</a> </li> <li> Ania: construct <a href="http://partsregistry.org/Part:BBa_K177014"> BBa_K177014</a> </li> <li> Monika: transform competent cells with <a href="http://partsregistry.org/Part:BBa_K1763004"> BBa_K1763004</a> , <a href="http://partsregistry.org/Part:BBa_S03473"> BBa_S03473</a> , <a href="http://partsregistry.org/Part:BBa_E0840"> BBa_E0840</a> , <a href="http://partsregistry.org/Part:BBa_J07037"> BBa_J07037</a> out of the distribution. </li> </ul> potential problems: get inv/llo/phoP/phoQ

Creating devices to test promoters in E. coli strains (devices BBa_K177024 and BBa_K177025) Franek Task: <ul> <li>transform competent cells with <a href="http://partsregistry.org/Part:BBa_I0500"> BBa_I0500 </a></li> </ul> Methods: <ul> <li>Resuspension of DNA from plate 1, 14N (<a href="http://partsregistry.org/Part:BBa_I0500"> BBa_I0500 </a>) with 15&micro;l of H2O</li> <li>Transformation of chemocompetent cells with 4&micro;l of <a href="http://partsregistry.org/Part:BBa_I0500"> BBa_I0500 </a> DNA solution</li> <li>Plating bacterias on LB medium supplemented with kanamycin</li> </ul> Results: <ul> <li>Will be determined tomorrow</li> </ul>

Jarek Task: <ul> <li>Preparation of bacterial cultures containing parts R0010, B0032 and C0051 in LB with ampicilin</li> </ul> Methods: <ul> <li>Concentration of ampicilin used for cultures was 100 ug/ml</li> </ul> Results: <ul> <li>The growth of the cultures will be observed on the next day</li> </ul>