Assembly of endosome detection operon

Marcin

Comment:

Result of following ligations was disappointing (no one bacterial colony on the plates):

• [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]

I examinated the effectiveness of DNA purification. The results reveal that the yield of used gel-out kit was surprisingly low. It forced me to prepare another enzymatic digestions.

Task 1: DNA digest to create following biobricks:

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

Methods:

• Constructs to digest:
  • [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
• Reaction mixture composition:

15 μl purified plasmid DNA product
1 μl enzyme 1 (Fermentas)
1 μl enzyme 2 (Fermentas) 
5 μl Buffer Tango (Fermentas)
28 μl MQ water

• Reactions were carried out 7 hour and subsequently they were inactivated via heating.