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Team:Warsaw/Calendar-Main/12 August 2009
From 2009.igem.org
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Assembly of endosomal detection operon
Marcin
Task 1:
- Digest of isolate plasmids to verify the success of isolation
Methods:
- Samples containing plasmids digested in 11.08.2009 were thawed and loaded into the agarose gel
Comment:
Results:
verification of the restriction patterns
Comment
All isolation were successful
Task 2:
- Isolate the crobox sequence from digested construct
Methods:
- After the digestion reaction mixture was loaded on the gel and electrophoretically separated
Results:
Elucidation of the digestion
Comment:
There was no sign of digested crobox sequence. Probably the concentration of the crobox DNA was low. To prepare digested sample it is necessary to use more concentrated solution of the crobox.
Task 3:
- Transformation of chemocompetent E. coli strain DH5α
Comment:
Due to obtain more amount previously cloned constructs the bacteria were transformed with followed constructs:
- p53 on pSB1A3 plasmid
- BBa_B0032+BBa_C0040 on pSB1A3 plasmid
- BBa_B0032+BBa_C0051 on pSB1A3 plasmid
- BBa_B0032+BBa_E0032 on pSB1A3 plasmid
Methods:
- Detailed protocol of transformation is described here.
Task 4:
- Restriction digest of biobricks
Comment:
Due to obtain set of biobricks which each of them contain RBS and particular coding sequence some of previously prepared constructs or biobricks were digested:
cro CDS on pKSII vector
Methods:
- Digest of BBa_R0080 using SpeI and PstI
- Reaction mixture composition:
20 μl purified plasmid DNA product 0.5 μl SpeI (Fermentas) 1 μl PstI (Fermentas) 5 μl Buffer Tango (Fermentas) 24 μl MQ water
- Digest of other constructs using PstI and XbaI
- 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
- All reaction were performed ~8 hours and both of them were subsequently inactivated via heating in 80°C for 20 minutes
- The reaction mixtures were loaded on the gel and DNA was electrophoretically separated. In the next step fragments of gel which contain appropriate DNA sequences were cut out and frozen
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