Team:LCG-UNAM-Mexico/Notebook/2008-July 02

LCG-UNAM-Mexico:Notebook/July_2   

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2008-07-24   WET LAB:  The cell colonies transformed with the biopart from Chiba(BBA_I729006), grew successfully.

Three colonies from each cage were plated(concentred and no concentred), in 5 mL of liquid LB. We run a with the PCR products.  Each track contains:  Molecular Marker  Product from part 1 in biopart BBa_I729006  Product from part 3 in biopart BBa_I729006</li>    Product from part 3 in biopart BBa_I729006 Making a mutation</li>  Product from RcnA</li>  Product from RcnA in strain RnA-(control)</li> </ol> In the first and second tracks there's no product, we will repeat this reactions. *Repeat PCR for Part1 and 2 in bioart BBa_I729006

<td class="subHeader" bgcolor="#99CC66" id="25">2008-07-25 <td class="bodyText"> WET LAB:  PCR We performed the PCR of the missing tubes Purification of PCR Product <img src="http://2008.igem.org/wiki/images/6/64/Gel_25Jul08.png" alt="Gel_25Jul08" width="400"/> <td class="subHeader" bgcolor="#99CC66" id="28">2008-07-28 <td class="bodyText"> WET LAB: Gel We run a gel in order to observe the PCR products of the 25th. <img src="http://2008.igem.org/wiki/images/2/2c/Gel_28Jul08_copy.png" alt="Gel_28Jul08" width="400" /> Restrictions(cuts) EcoR1-Up/Bam-Lowe          Parte1  (10 μl of PCR sample)

Xba1-Up/Pst1-Low              Parte3-N  (8 μl of PCR sample)

Xba1-Up/Pst1-Low              Parte3-M (5 μl of PCR sample)

Xba1-Up/HindIII-Lower       RcnA (5 μl of PCR sample)

The overnight reaction was at 37ºC. *BSA(Bovine Serum Albumin)

<td class="subHeader" bgcolor="#99CC66" id="29">2008-07-29 <td class="bodyText"> WET LAB: Cultures From each one of the samples cultured on 28th,we chose 5 colonies and we plated in a 1 mL LB tube and we incubate it for 6 hrs. Our neative control was just LB. Bioparts ligation in pRK415 and pBB Transformation We transformed the product of the ligation and plated it in cages with X-gal. With he purpose of save X-gal we chose to add it not in the LB-agar, but joint to the plated culture. In order to do this, we dilute 20 mL of X-Gal in 1mL of N-N-dimethylformamide. And from this mix we plated 25μl in each cage. For the transformation we used two controls( one without plasmid and one without cell). When plating we excluded the control without cell and plated the DH5 cells without transformation in a Tc Cage and another of Gm. Plasmid Extraction We extract plasmid from te plated colonies. All the tubes pass the night with 1mL of ethanol at 100%. Biopart cI  The missing biopart(cI), was cultured in 5mL of LB with 2.5 μl of ampicillin(50%). It was also plated in 5mL of LB without antibiotic and was plated in agar with ampicillin at 100%

<td class="subHeader" bgcolor="#99CC66" id="30">2008-07-30 <td class="bodyText"> WET LAB: Plasmid extraction We finished the extraction of the plasmid, started on 29th.

We extracted the plasmid holding the biopart cI. Gel We run a Gel with the 15 samples of the plasmid pHET containing RcnA, part 3(normal) or part 3 (mut) and the three extraction plasmid samples containing the biopart cI+LVA

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