Team:Warsaw/Calendar-Main/29 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 20 hours in 16 &deg;C.

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

Comment:

All performed ligation were unsuccessful. I am afraid that the effectivity of our gel-out kit may be lower than is expected. Someone must find it out.

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 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:
 * Will be seen tomorrow

Isolation of BBa_J63010 from 2009 Kit
 Monika 

Task: Control digestion of BBa_J63010

Methods 5&mu;l plasmid solution 0,3&mu;l EcoRI (Fermentas) 0,3&mu;l PstI (Fermentas) 2&mu;l Buffer Orange (Fermentas) 12,4 μl MQ water
 * First reaction mixture composition:

5&mu;l plasmid solution 0,3&mu;l PvuII (Fermentas) 2&mu;l Buffer Green (Fermentas) 12,7 μl MQ water
 * Second reaction mixture composition:


 * Digestion in 37&deg;C for 2,5h


 * Results:

Cloning of the cro-box into the pSB1A3 plasmid Kamil

Tasks:  Bacteria transformation 

Methods:  A batch of chemocompetent bacteria was transformed with the ligated plasmid and incubated in 37&deg;C overnight on agarose plates supplemented with ampicilin. 