Assembly of endosomal detection operon

Marcin

Task 1:

• Digest of BBa_B0032

Methods:

• Digest of BBa_B0032 using SpeI and PstI
• Reaction mixture composition:

  10 μl purified plasmid DNA product
  0.5 μl SpeI (Fermentas)
  1 μl PstI (Fermentas)
  5 μl Buffer Tango (Fermentas)
  34 μl MQ water

• The reaction was performed overnight (~12 hours) and it was subsequently inactivated via heating in 80°C for 20 minutes
• After inactivation sample was electrophoretically separated on the agarose gel:


Digested product
on the gel

Task 2:

• Purification of digested product via gel-out

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
• Quantification of amount of biobrick DNA after restriction digest:

1 μl of the digest mixture was diluted to 10 μl and loaded into the gel.

Task 3:

• Ligations of biobricks

List of designed biobricks:

1. BBa_B0032+BBa_c0040
2. BBa_B0032+BBa_C0051
3. BBa_B0032+BBa_E0032

Methods:

• Ligation mixture composition:

  3 μl digested plasmid with RBS
  3 μl digested CDS
  5 μl ligation buffer (Fermentas, PEG4000 have been added previously)
  1 μl ligase T4
  13 μl MQ water

• Duration of ligation was about 12 hours

Kama

Notes

• DNA disapeared, probably during inactivation