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:

• Reaction mixtures composition:

45 μl mixture of insert and vector DNA (both are purified at the same probe)
1.5 μl T4 ligase (Fermentas)
5 μl Ligase Buffer (Fermentas)

• Ligation was carried out 20 hours in 16 °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:

• PCR mixture:

 5μl Pfu polymerase buffer
 1μl forward primer and 1μl reverse primer
 2μl dNTPs (10 mM)
 2,5μl Pfu turbo polymerase (EURX)
 2μl template DNA from Salmonella enterica typhimurium LT2
 The solution was topped up with H2O to 10μl
 
 contol - no template DNA from Salmonella enterica typhimurium LT2

• PCR conditions:

   1. 3min 95°C
   2. 30s 95°C
   3. 35s 58°C
   4. 2min 10 s 72°C
   5. go to step 2, 2 times
   6. 30s 95°C
   7. 30s 68°C
   8. 2min 10s 72°C
   9. go to step 6, 28 times
   10. 10min 72°C 
   11. forever 4°C

Results of PCR:

• Will be seen tomorrow

Isolation of BBa_J63010 from 2009 Kit

Task: Control digestion of BBa_J63010

Methods

• First reaction mixture composition:

     5μl plasmid solution
     0,3μl EcoRI (Fermentas)
     0,3μl PstI (Fermentas) 
     2μl Buffer Orange (Fermentas)
     12,4 μl MQ water

• Second reaction mixture composition:

     5μl plasmid solution
     0,3μl PvuII (Fermentas) 
     2μl Buffer Green (Fermentas)
     12,7 μl MQ water

• Digestion in 37°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°C overnight on agarose plates supplemented with ampicilin.