Team:Warsaw/Calendar-Main/24 September 2009

Assembly of endosome detection operon Marcin

Comment:

Result of following ligations was disappointing (no one bacterial colony on the plates):


 * BBa_K177035 with BBa_K177036 to obtain BBa_K177037
 * BBa_K177035 with BBa_K177043 to obtain BBa_K177044

I examinated the effectiveness of DNA purification. The results reveal that the yield of used gel-out kit was surprisingly low. It forced me to prepare another enzymatic digestions.

Task 1: DNA digest to create following biobricks:
 * BBa_K177037
 * BBa_K177044

Methods: 15 μl purified plasmid DNA product 1 μl enzyme 1 (Fermentas) 1 μl enzyme 2 (Fermentas) 5 μl Buffer Tango (Fermentas) 28 μl MQ water
 * Constructs to digest:
 * BBa_K177035 - PstI, SpeI
 * BBa_K177035 - PstI, XbaI
 * BBa_K177036 - PstI, SpeI
 * BBa_K177044 - PstI, XbaI
 * Reaction mixture composition:
 * Reactions were carried out 7 hour and subsequently they were inactivated via heating.

PCR of phoP

Monika

Task:
 * amplification of phoP (675 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 contol - no template DNA from Salmonella enterica typhimurium LT2
 * 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 of PCR:
 * There were no products of phoP amplification


 * There were no products of phoQ amplification

Comment:
 * We suppose there were some problems with polymerase and PCR programme was wrongly chosen

Isolation of BBa_J63010 from 2009 Kit

Monika

Tasks:


 * Isolate BioBrick BBa_J63010 from 2009 Kit Plate 1

Methods:
 * Alkaline lysis with A&A Biotechnology Kit, procedure described here

Monika

Task:
 * amplification of phoP (675 bp) and 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 contol - no template DNA from Salmonella enterica typhimurium LT2
 * PCR mixture:

1. 3min 95&deg;C 2. 30s 95&deg;C 3. 35s 59&deg;C 4. 2min 72&deg;C 5. go to step 2, 2 times 6. 30s 95&deg;C 7. 2min 40s 72&deg;C 8. go to step 6, 28 times 9. 10min 72&deg;C 10. forever 4&deg;C
 * PCR conditions:

Results of PCR:

Cloning of the cro-box into the pSB1A3 plasmid Kamil

Tasks:  Selection of the colonies 

Methods:  Additional colonies were selected and transferred to a fresh agarose plate, liquid cultures were established. The plate and the liquid cultures were incubated overnight in 37&deg;C 