Team:Warsaw/Calendar-Main/28 September 2009

Assembly of endosome detection operon Marcin

Task 1: Prepare the following ligations:
 * BBa_K177035 with BBa_K177036 to obtain BBa_K177037
 * BBa_K177035 with BBa_K177043 to obtain BBa_K177044

Methods: 45 &mu;l mixture of insert and vector DNA (both are purified at the same probe) 1.5 &mu;l T4 ligase (Fermentas) 5 &mu;l Ligase Buffer (Fermentas)
 * Reaction mixtures composition:
 * Ligation was carried out 6 hours in 16 &deg;C. Subsequently both mixtures were divided up two portions. In the case of each reaction one portion was thermally inactivated and the latter one was unremoved.

Task 2: 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

PCR of phoP and phoQ

Monika

Task:
 * amplification of phoP (675 bp) and phoQ (1464 bp)

Methods: 1&mu;l Pfu polymerase buffer 0,2&mu;l forward primer and 1&mu;l reverse primer 0,4&mu;l dNTPs (10 mM) 0,5&mu;l Pfu turbo polymerase (EURX) 0,4&mu;l template DNA from Salmonella enterica typhimurium LT2 The solution was topped up with H2O to 10&mu;l contol - no template DNA from Salmonella enterica typhimurium LT2
 * PCR mixture:

1. 3min 95&deg;C 2. 30s 95&deg;C 3. 35s 58&deg;C 4. 2min 10 s 72&deg;C 5. go to step 2, 2 times 6. 30s 95&deg;C 7. 30s 68&deg;C 8. 2min 10s 72&deg;C 9. go to step 6, 28 times 10. 10min 72&deg;C 11. forever 4&deg;C
 * PCR conditions:

Results of PCR:

Bistable switch testing
 Monika 

Task: Control digestion of bistable switch

Methods 6&mu;l plasmid solution 0,3&mu;l HindIII (Fermentas) 0,3&mu;l NheI (Fermentas) 2&mu;l Buffer Tango (Fermentas) 11,4 μl MQ water
 * Reaction mixture composition:


 * Digestion in 37&deg;C for 2 h

Results
 * Will be seen tomorrow

Cloning of the cro-box into the pSB1A3 plasmid Kamil

Tasks:  Cro-box preparation Ligation 

Methods:  Two short oligonucleotides were annealed to form a complete double stranded cro-box sequence with XbaI and PstI sticky ends. Equal concentrations of both oligonucleotides were mixed together, heated to 95&deg;C for 15 min. and left to cool. The pSB1A3 plasmid was digested with XbaI and PstI and the resulting backbone was isolated (all as described previously). The ligation was carried out in 18&deg;C for 4h. 