Team:Warsaw/Calendar-Main/19 July 2009
From 2009.igem.org
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:
- Purification of digested product via gel-out
- 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:
- Ligations of biobricks
- 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
- DNA disapeared, probably during inactivation
Digested product
on the gel
on the gel
Task 2:
Methods:
1 μl of the digest mixture was diluted to 10 μl and loaded into the gel.
Task 3:
List of designed biobricks:
Methods:
Kama
Notes
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