Team:Warsaw/Calendar-Main/23 September 2009

Gradient PCR PhoP/PhoQ Kama

Tasks:  Amplification of phoP/phoQ 

Methods:  PCR mixture's composition: 1&mu;l Pfu buffer (EURX) 0,25&mu;l primer phoPF 0,25&mu;l primer phoQR 0,5&mu;l dNTPs (10 mM) 0,25&mu;l Pfu turbo polymerase (EURX) 0,5&mu;l template DNA from Salmonella (unknown strain) 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 55-65&deg;C, 5min 10s 72&deg;C)x35 10min 72&deg;C ~ 7&deg;C   Electrophoretic separation on 1% agarose gel 

Results: <img src="http://2009.igem.org/wiki/images/3/3b/2009_09_24_phoP_phoQ_new_opisane.JPG"/>  Gel (from left)</li> </ul> <ol> M - GeneRuler DNA Ladder Mix #SM0333 (Fermentas) control -</li> 3-5 - samples (annealing temperature increases to from the left to the right)</li> </ol> Notes:PhoP/PhoQ was succesfully amplified  </b></li> </ul>

Insertion of the pho gene into the pSB plasmid Kama  Isolation of plasmid containing an insert was performed with the <a href=http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf>A&A plasmid mini kit</a></li> </ul> Notes  </li> </ul>

Isolation of BBa_J63010 from 2009 Kit

Monika

Tasks:


 * Isolate BioBrick BBa_J63010 from 2009 Kit Plate 1

Methods:
 * 2009 Kit: resuspension of DNA from selected wells with 15ul of H2O
 * Transformation of chemocompetent cells with 3ul of DNA solution - procedure destribed here
 * Planting on LB-agar medium supplemented with ampicilin

Results:
 * Will be determined tomorrow.

PCR of phoQ

Monika

Task:
 * amplification of phoQ (1464 bp)

Methods: 5&mu;l Pfu polymerase buffer 1&mu;l forward primer and 1&mu;l reverse primer 2&mu;l dNTPs (10 mM) 2,5&mu;l Pfu turbo polymerase (EURX) 2&mu;l template DNA from Salmonella enterica typhimurium LT2 The solution was topped up with H2O to 50&mu;l
 * PCR mixture:

1. 5min 95&deg;C 2. 30s 95&deg;C 3. 2min 72&deg;C 4. go to step 2 x30 5. 10min 72&deg;C 6. forever 4&deg;C
 * PCR conditions:

Results:
 * Will be seen tomorow

Assembly of endosome detection operon Marcin

Task 1: Gel-out of the following biobricks (digested in 22.09.09):


 * BBa_K177035 - PstI, SpeI
 * BBa_K177035 - PstI, XbaI
 * BBa_K177036 - PstI, SpeI
 * BBa_K177044 - PstI, XbaI

Methods:

All gel-outs were performed using the A&A kit according to the manual

Task 2: Prepare the following ligations:
 * BBa_K177035 with BBa_K177036 to obtain BBa_K177037
 * BBa_K177035 with BBa_K177043 to obtain BBa_K177044

Methods 11 &mu;l insert 7 &mu;l vector 1 &mu;l T4 ligase (Fermentas) 2.4 &mu;l Buffer Tango (Fermentas) 2.5 &mu;l dNTPs mixture (EurX, concentration 5 mM)
 * Reaction mixtures composition:
 * Ligation was carried out 10 hours in 16 &deg;C. Subsequently mixtures were thermally inactivated

Task 3: Transformation of TOP10 chemocompetent bacteria with following constructs:


 * BBa_K177044
 * BBa_K177037

Methods:
 * Ligation mixture was thermally inactivated
 * Detailed protocol of transformation is described here