Gradient PCR PhoP/PhoQ

Kama

Tasks:

• Amplification of phoP/phoQ

Methods:

• PCR mixture's composition:

  1μl Pfu buffer (EURX) 
  0,25μl primer phoPF
  0,25μl primer phoQR 
  0,5μl dNTPs (10 mM)
  0,25μl Pfu turbo polymerase (EURX)
  0,5μl template DNA from Salmonella (unknown strain)
  0,75μl DMSO 
  The solution was topped up with H2O to 10μl.

• PCR programs:

pho

4min 95°C 
 (30s 95°C, 1min 55-65°C, 5min 10s 72°C)x35
 10min 72°C 
 ~ 7°C

• Electrophoretic separation on 1% agarose gel

Results:

Insertion of the pho gene into the pSB plasmid

Kama

• Isolation of plasmid containing an insert was performed with the A&A plasmid mini kit

Notes

Isolation of BBa_J63010 from 2009 Kit

Monika

Tasks:

• Isolate BioBrick [http://partsregistry.org/Part:BBa_J63010 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:

• 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 50μl

• PCR conditions:

   1. 5min 95°C
   2. 30s 95°C
   3. 2min 72°C
   4. go to step 2 x30 
   5. 10min 72°C 
   6. forever 4°C

Results:

• Will be seen tomorow

Assembly of endosome detection operon

Marcin

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

• [http://partsregistry.org/Part:BBa_K177035BBa_K177035] - PstI, SpeI
• [http://partsregistry.org/Part:BBa_K177035BBa_K177035] - PstI, XbaI
• [http://partsregistry.org/Part:BBa_K177036BBa_K177036] - PstI, SpeI
• [http://partsregistry.org/Part:BBa_K177044BBa_K177044] - PstI, XbaI

Methods:

All gel-outs were performed using the A&A kit according to the [http://aabiot.com/products/dna_purification/dna_fragments/gel_out/protocol_gel_out.pdf manual]

Task 2: Prepare the following ligations:

• [http://partsregistry.org/Part:BBa_K177035BBa_K177035] with [http://partsregistry.org/Part:BBa_K177036BBa_K177036] to obtain [http://partsregistry.org/Part:BBa_K177037BBa_K177037]
• [http://partsregistry.org/Part:BBa_K177035BBa_K177035] with [http://partsregistry.org/Part:BBa_K177043BBa_K177043] to obtain [http://partsregistry.org/Part:BBa_K177044BBa_K177044]

Methods

• Reaction mixtures composition:

11 μl insert
7 μl vector
1 μl T4 ligase (Fermentas)
2.4 μl Buffer Tango (Fermentas)
2.5 μl dNTPs mixture (EurX, concentration 5 mM)

• Ligation was carried out 10 hours in 16 °C. Subsequently mixtures were thermally inactivated

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

• [http://partsregistry.org/Part:BBa_K177044BBa_K177044]
• [http://partsregistry.org/Part:BBa_K177037BBa_K177037]

Methods:

• Ligation mixture was thermally inactivated
• Detailed protocol of transformation is described here