Team:Warsaw/Calendar-Main/9 July 2009

Isolation of BioBricks from 2008 and 2009 Kit Plates
Monika

Tasks:


 * Isolate plasmids containing the biobricks


 * 1) GFP coding device switched on by IPTG - BBa_I763004  from 2008 Kit Plate 1017 well G5
 * 2) promoter lambda (cI regulated) with RFP reporter BBa_I763007  from 2009 Kit Plate 1 well 15J
 * 3) GFP generator - BBa_E0840 from 2009 Kit Plate 1 well 12O
 * 4) GFP generator - BBa_J07037 from 2009 Kit Plate 1 well 12O

Methods: here)
 * 2009 Kit: resuspension of DNA from selected wells with 15ul of H2O
 * 2008 Kit: isolation of DNA from selected wells (two punched paper spots) with 8ul of TE (prodedure described
 * Transformation of chemocompetent cells (prepared by Franek and Ania) with 3ul of DNA solution
 * Planting on LB-agar medium supplemented with apropriate antibiotic

Results
 * Will be determined tomorrow.

Creating devices to test promoters in E. coli strains (devices BBa_K177024 and BBa_K177025)
Franek

Tasks:


 * Alkaline lysis of the plasmid containing BBa_I0500


 * Transform competent cells with BBa_B0024

Methods:
 * Second attempt to transform competent cells with BBa_I0500


 * Plates with BBa_I0500 were empty, therefore once again transformation of chemocompetent cells was performed, but this time 8&micro;l of BBa_I0500 DNA solution was used


 * Plating bacterias with BBa_I0500 on LB medium supplemented with kanamycin


 * Resuspension of DNA from plate 1, 2C (BBa_B0024) with 15&micro;l of H2O


 * Transformation of chemocompetent cells with 4&micro;l of BBa_B0024 DNA solution

Results:
 * Plating bacterias with BBa_B0024 on LB medium supplemented with ampicillin


 * Will be determined tomorrow

Jarek Task:  Isolation of plasmid containing parts from liquid cultures Digestion of acquired samples with restriction endonucleases Electrophoretic separation of digested samples Isolation of samples from agarose gel  Methods:  Plasmid DNA was isolated with A&A "Plasmid Mini" kit, the DNA concentration was measured with nanodrop For digestion 1 ul of PstI/SpeI () or PstI/XbaI enzymes and 2 ul of 1xTango buffer were used. Digestion was held for 3 hours. After digestion samples were separated due to elecrophoresis in 0,8 agarose gel in TBE buffer</li> Samples were isolated from gel with A&A "Gel-out" kit</li> </ul> Results:  </li> </ul>

Cloning of p53 coding sequence Marcin Comment: Because of completely degradation of PCR product there is urgent need to amplified the p53 coding sequence. I think the original plasmid with the p53 is preserved and I'm about to repeat the PCR reaction using the plasmid as the template Task:  Prepare PCR reaction to amplified p53 coding sequence.</li> </ul> Methods:  PCR mixture composition:</li> </ul> 1. proper mixture: 0.25 ul primer 1 (50 nM; Oligo.pl) 0.25 ul primer 2 (50 nM; Oligo.pl) 1.5 ul dNTPs (20 uM ;Fermentas) 0.5 ul Pfu turbo polymerase (KNGiE) 2.5 ul Pfu Turbo Buffer (Fermentas) 2.5 ul MgSO4 (20 uM; Fermentas) 1 ul DNA template, 16.5 ul MQ water 2. Negative control: the same as previously described proper mixture, the only distinction is lack of the DNA template.

 Program:</li> </ul> p53 1. 98&deg;C - hold 2. 98&deg;C - 2 minutes 3. 98&deg;C - 15 sec 4. 64&deg;C - 30 sec 5. 68&deg;C - 2 minutes go to 2 until number of cycles=35 6. 68&deg;C - 10 minutes 7. 4&deg;C - hold  Results:</li> </ul> After PCR the reaction was divided to 3 portion and loaded into the 1% agarose gel to photograph and subsequently gel-out the sample. <img src="http://2009.igem.org/wiki/images/5/5e/PCR_p53_09_07_09.jpg" align="center" width="45%" heigth="45%"> PCR products loaded into gel Used DNA Ladder: DNA Ruler (Fermentas), 5 ul Comment: The effectiveness of PCR reaction was surprisingly low, probably due to degradation of the DNA template. Reamplification of the p53 sequence using the purificated DNA sample from the gel seems to be a good idea. Of course sometimes reamplification lead to obtain partially degraded sequences but I think I should try this method.

Reamplification of p53 Task 1:  Purification of amplified p53 via gel-out procedure</li> </ul> Methods:  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> Task 2: <ul> <li>Prepare another PCR reaction to reamplified p53 coding sequence using the product of previous PCR as a template</li>. Methods: </ul> <ul> <li>PCR mixture composition:</li> </ul> 1. proper mixture: 0.25 ul primer 1 (50 nM; Oligo.pl) 0.25 ul primer 2 (50 nM; Oligo.pl) 1.5 ul dNTPs (20 uM; Fermentas) 0.5 ul Pfu turbo polymerase (KNGiE) 2.5 ul Pfu Turbo Buffer (Fermentas) 2.5 ul MgSO4 (20 uM; Fermentas) 2 ul DNA template 15.5 ul MQ water 2. Negative control: the same as previously described proper mixture, the only distinction is lack of the DNA template. <ul> <li>Program:</li> </ul> p53, looked above to see details <ul> <li>Results:</li> After PCR the reaction was divided to 2 portion and loaded into the 1% agarose gel to photograph using the UV transiluminator Used DNA Ladder: DNA Ruler (Fermentas), 5 ul <img src="http://2009.igem.org/wiki/images/b/b4/PCR_reamplifikacja_p53_09_07_09_.png" align="center"> 1,2 - PCR reaction, sample was divided to 2 portion Comment: The reaction was unsuccessful, obtained products are not specific, besides they are too short (below 500 bp).