Assembly of endosomal detection operon

Marcin

Task 1:

• Gel-out of following sequences from the pSB1A3 plasmid:
  • p53 CDS
  • BBa_B0032+BBa_C0040
  • BBa_R0080
  • BBa_E0032

Methods:

• Gel-outs were prepared according to manufacturer's manual

Results:

Comment:

Some purification were unsuccessful because time required to efficacious activation of the columns is, in fact longer than few minutes (which is not depicted in the manual). It is obligated to repeat digestion and gel-out for the samples which were not isolated

Task 2:

• Ligation of following constructs:
• p53 CDS to BBa_B0032 on pSB1A3 plasmid
• BBa_C0040 to BBa_R0080 on pSB1A3 plasmid

Methods:

• Ligation mix:

1.0 μl - T4 ligase
2.5 μl - Tango buffer (Fermentas, 10x)
3.0 μl - dNTPs (EURx, 5μlM)
6.0 μl - digested vector
12.5 μl - digested insert

• Ligation time - overnight

Task 3:

• Prepare the bacterial cultures for isolation of following plasmids:
  • BBa_E0022 with BBa_B0032 on pSB1A3 plasmid
  • backbone plasmid pSB1A3

Task 4:

• Transformation of chemocompetent E. coli strain TOP10 to reveal the quality of prepared bacteria

Plasmid to transform: pSB1A3

Methods:

• Detailed protocol of transformation is described here.