Team:Warsaw/Calendar-Main/14 August 2009

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Cloning Bax into pBS plasmid

Justyna

Task 1:

  • Gel-out Bax PCR product

Methods:

  • Bax PCR products were isolated from agarose gel using A&A Gel-Out kit.


Task 2:

  • Digestion and gel-out of pBS plasmid

Methods:

  • Digestion of isolated plasmids by XbaI and SpeI
  • Reaction mixture composition: 10.0 μl purified plasmid DNA product, 0.5 μl XbaI (Fermentas),0.5 μl SpeI (Fermentas), 2 μl Buffer Orange (Fermentas), 7.0 μl MQ water
  • The digestion was performed overnight and it was subsequently inactivated via heating in 80°C for 20 minutes.
  • In the next step reaction mixtures were loaded into the agarose gel to analize restriction pattern of the plasmids and isolated using A&A Gel-Out kit

Task 3:

  • Ligation of Bax and pBS

Methods:

  • Ligation mix:
    • 1.0 μl - T4 ligase
2.5 μl - Tango buffer (Fermentas, 10x)
2.0 μl - dNTPs (EURx, 5μlM)
5.0 μl - digested pSB (~100 ng)
14.5 μl - Bax (~ 105 ng)
in 25.0 μll
  • Ligation time - overnight


Task 4:

  • Insertion of Bax-pBS plasmid into bacteria

Methods:

  • Previously prepared chemicompetent bacteria were used.





Assembly of endosomal detection operon

Marcin


Task 1:

  • Isolate the plasmid prepared in 13.08.09

Methods:

  • Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here

Task 2:

Methods:

  • Reaction mixture composition:
    •  
20 μl purified plasmid DNA product
1 μl XbaI (Fermentas) or 0,5 μl SpeI (Fermentas) in the case of R0080
1 μl PstI (Fermentas)
5 μl Buffer Tango (Fermentas)
24 μl MQ water

Task 3:

  • Gel-out of digest sequences described in Task 1

Methods:

  • Fragments of agarose gel were carefully cut out and subsequently frozen in -20°C./gel_out/protocol_gel_out.pdf">here

Comment:

Restriction digests of samples containing BBa_E0022 and BBa_B0032+BBa_C0040 were unsuccessful, probably the probes were contaminared. Because of this fact I decided to repeat the digest.


Task 4:

  • Another digest of E0022 and C0040+B0032

Methods:

  • Reaction mixture composition:
    •  
20 μl purified plasmid DNA product
1 μl XbaI (Fermentas)
1 μl PstI (Fermentas)
5 μl Buffer Tango (Fermentas)
24 μl MQ water

Task 5:

  • Transformation of chemocompetent E. coli strain DH5&alpha

Constructs to transform:

Methods:

  • Ligation prepared in 08.08.09 was stopped via thermal inactivation in 80°C for 20 minutes
  • Detailed protocol of transformation is described here.

Task 6:

  • Prepare chemocompetent TOP10 bacteria strain (I worked with Kuba)

Methods:

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