Team:Warsaw/Calendar-Main/14 August 2009
From 2009.igem.org
(Difference between revisions)
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<li>Ligation of Bax and pBS</li></ul> | <li>Ligation of Bax and pBS</li></ul> | ||
<p>Methods:</p><ul> | <p>Methods:</p><ul> | ||
+ | <li>Ligation mix:</li> | ||
+ | <pre> | ||
+ | 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 | ||
+ | </pre> | ||
<li>The full description of ligation is here <a href=...></a></li></ul> | <li>The full description of ligation is here <a href=...></a></li></ul> | ||
<br/> | <br/> |
Revision as of 23:49, 15 August 2009
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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