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Team:Warsaw/Calendar-Main/14 August 2009
From 2009.igem.org
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Cloning Bax into pSB1A3 plasmid
Justyna
Task 1:
- Gel-out Bax PCR product
Methods:
- Bax PCR products were isolated from agarose gel using A&A Gel-Out kit.
Task 2:
- Digestion and gel-out of pSB1A3 plasmid
Methods:
- Digestion of isolated plasmids by XbaI and SpeI
- Reaction mixture composition: 10.0 μl purified plasmid DNA product, 0.5 μl XbaI (Fermentas),0.5 μl SpeI (Fermentas), 2 μl Buffer Orange (Fermentas), 7.0 μl MQ water
- The digestion was performed overnight 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 and isolated using A&A Gel-Out kit
Task 3:
- Ligation of Bax and pSB1A3
Methods:
- Ligation mix:
1.0 μl - T4 ligase 2.5 μl - Tango buffer (Fermentas, 10x) 2.0 μl - dNTPs (EURx, 5μlM) 5.0 μl - digested pSB1A3 (~100 ng) 14.5 μl - Bax (~ 105 ng) in 25.0 μll
Task 4:
- Insertion of Bax-pSB1A3 plasmid into bacteria
Methods:
- Previously prepared chemicompetent bacteria were used.
Assembly of endosomal detection operon
Marcin
Task 1:
- Isolate the plasmid prepared in 13.08.09
Methods:
- Plasmids were isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here
Task 2:
- Restriction digest of following sequences from the pSB1A3 plasmid:
Methods:
- Reaction mixture composition:
20 μl purified plasmid DNA product 1 μl XbaI (Fermentas) or 0,5 μl SpeI (Fermentas) in the case of R0080 1 μl PstI (Fermentas) 5 μl Buffer Tango (Fermentas) 24 μl MQ water
Task 3:
- Gel-out of digest sequences described in Task 1
Methods:
- Fragments of agarose gel were carefully cut out and subsequently frozen in -20°C>
Results
Verification of the digestions
Comment:
Restriction digests of samples containing BBa_E0022 and BBa_B0032+BBa_C0040 were unsuccessful, probably the probes were contaminared. Because of this fact I decided to repeat the digest.
Task 4:
- Another digest of E0022 and C0040+B0032
Methods:
- 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
Task 5:
- Transformation of chemocompetent E. coli strain DH5&alpha
Constructs to transform:
Methods:
- Ligation prepared in 08.08.09 was stopped via thermal inactivation in 80°C for 20 minutes
- Detailed protocol of transformation is described here.
Task 6:
- Prepare chemocompetent TOP10 bacteria strain (I worked with Kuba)
Methods:
- Detailed protocol is described here
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