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.
• The full description of the procedure is here

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:

• Restriction digest of following sequences from the pSB1A3 plasmid:
  • p53 CDS
  • BBa_B0032+BBa_C0040
  • BBa_R0080
  • BBa_B0032+BBa_E0022

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 in the next step DNA was extracted from the gel using the A&A gel-out kit. Detailed procedure is described here

Comment:

The concentration of DNA is samples containing BBa_E0022 and BBa_B0032+BBa_C0040 was surprisingly low. Because of this fact I decided to repeat the digest.

Task 4:

• Another digest of E0032 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