Team:Warsaw/Calendar-Main/14 August 2009
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Revision as of 23:49, 15 August 2009 by Justyna lesiak (Talk | contribs)
Cloning Bax into pBS 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 pBS 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 pBS
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 pSB (~100 ng) 14.5 μl - Bax (~ 105 ng) in 25.0 μll
Task 4:
- Insertion of Bax-pBS plasmid into bacteria
Methods:
- Previously prepared chemicompetent bacteria were used.
- The full description of the procedure is here
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