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Team:Warsaw/Calendar-Main/6 August 2009
From 2009.igem.org
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Cloning of p53 coding sequence
Marcin
Task 1:
- Isolate the plasmid containing p53 CDS from bacterial cultures and verify the effectivity of the isolation via gel electrophoresis
- Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here
Methods:
Results:
Evaluation of the isolation yield
Samples denoted as A-C are additional isolations of pSB1A3 plasmid
Samples denoted as A-C are additional isolations of pSB1A3 plasmid
Task 2:
- Digest of isolate plasmids with ligated p53 CDS to verify the success of ligation
Methods:
- Digest of isolate plasmids using EcoRI and PstI
- Reaction mixture composition:
0.5 μl purified plasmid DNA product 0.5 μl EcoRI (Fermentas),0.5 μl PstI (Fermentas) 2 μl Buffer Orange (Fermentas) 16.5 μl MQ water
- The digest was performed three hours and it was subsequently inactivated via heating in 80°C for 20 minutes.
- In the next step reaction mixtures were loaded into the agarose gel to analyse restriction pattern of the plasmids
Results:
Verification of the restriction patterns
Comment
Four samples showed expected restriction pattern - they contain insert but it has no been determinated whether the orientation of the insert is correct. It is expected to perform another digest using XbaI and PstI to confirm the correctness of ligation
Task 2:
- Second digest in order to determinate the directiom of insert
Methods:
- Digest of isolate plasmids using XbaI and PstI
- Reaction mixture composition:
0.5 μl purified plasmid DNA product 0.5 μl XbaI (Fermentas) 0.5 μl PstI (Fermentas) 2 μl Buffer Tango (Fermentas) 16.5 μl MQ water
- The digest was performed three hours and it was subsequently inactivated via heating in 80°C for 20 minutes.
- In the next step reaction mixtures were loaded into the agarose gel to analize restriction pattern of the plasmids
Results:
Verification of the restriction patterns
Comment
Only one sample has insert cloned in proper direction - sample nr 3
Assembly of endosomal detection operon
Marcin
Task 1:
- Isolate the plasmid containing BBa_B0032 and BBa_C0040 from bacterial cultures
- Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here
Methods:
Task 2:
- Digest of isolate plasmids with ligated biobricks to verify the success of ligation using XbaI and PstI<
Methods:
-
Reaction mixture composition:
0.5 μl purified plasmid DNA product 0.5 μl XbaI (Fermentas) 0.5 μl PstI (Fermentas) 2 μl Buffer Tango (Fermentas) 16.5 μl MQ water
- The reaction was performed three hours and it was subsequently inactivated via heating in 80°C for 20 minutes.
- After the inactivation samples were frozen in -20°c
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