Team:Valencia/Transforming Coccus
From 2012.igem.org
(Difference between revisions)
(Created page with "{{:Team:Valencia/menu}} {{:Team:Valencia/fondoweb}} <html> <div id="Titulos"> <br><br> Transforming: <i>Synechococcus elongatus</i> PCC7942 </div> <div id="HomeCenterCenter"><...") |
|||
Line 27: | Line 27: | ||
<li>Resuspend the cell pellet in 10 mL of 10mM Nacl and harvest by centrifugation for 10 min at 6000gx.</li> | <li>Resuspend the cell pellet in 10 mL of 10mM Nacl and harvest by centrifugation for 10 min at 6000gx.</li> | ||
<li>Resuspend the cell pellet in 0.3 mL of BG-11M and transfer to a microcentrifuge tube.</li> | <li>Resuspend the cell pellet in 0.3 mL of BG-11M and transfer to a microcentrifuge tube.</li> | ||
- | <li>To each 0.3 mL, add between 50 ng and 2 | + | <li>To each 0.3 mL, add between 50 ng and 2 μg of DNA (typically, we use 1-2 μl from a preparation of 100-200ng/μl).</li> |
<li>Wrap the tubes in aluminum foil the shield the cells from light and incubate them overnight at 30<sup>o</sup>C with gentle agitation.</li> | <li>Wrap the tubes in aluminum foil the shield the cells from light and incubate them overnight at 30<sup>o</sup>C with gentle agitation.</li> | ||
<li>Plate the entire 0.3-mL cell suspension on a BG-11 Medium plate containing the appropiate selective medium.</li> | <li>Plate the entire 0.3-mL cell suspension on a BG-11 Medium plate containing the appropiate selective medium.</li> | ||
- | <li>Incubate the plates at | + | <li>Incubate the plates at 30ºC in constant light for aprox. 5 d until single colonies appear.</li> |
<li>Restreak isolated colonies that have the appropiate phenotypes, maintaining the selection to favor complete segregation of mutant cyanobacterial chromosomes.</li> | <li>Restreak isolated colonies that have the appropiate phenotypes, maintaining the selection to favor complete segregation of mutant cyanobacterial chromosomes.</li> | ||
<li>Grow mutant clones in 100ml of BG-11.</li> | <li>Grow mutant clones in 100ml of BG-11.</li> |
Latest revision as of 02:23, 27 September 2012
Transforming: Synechococcus elongatus PCC7942
Reagents and Materials
- Synechococcus elongatus PCC7942
- Plates
- 0’3 mL BG-11
- 10 mL of 10mM Nacl
- Light
Protocol
- Grow 100 ml of S. elongatus.
- Harvest 15 ml of cyanobacterial cells by centrifugation for 10 min at 6000gx.
- Resuspend the cell pellet in 10 mL of 10mM Nacl and harvest by centrifugation for 10 min at 6000gx.
- Resuspend the cell pellet in 0.3 mL of BG-11M and transfer to a microcentrifuge tube.
- To each 0.3 mL, add between 50 ng and 2 μg of DNA (typically, we use 1-2 μl from a preparation of 100-200ng/μl).
- Wrap the tubes in aluminum foil the shield the cells from light and incubate them overnight at 30oC with gentle agitation.
- Plate the entire 0.3-mL cell suspension on a BG-11 Medium plate containing the appropiate selective medium.
- Incubate the plates at 30ºC in constant light for aprox. 5 d until single colonies appear.
- Restreak isolated colonies that have the appropiate phenotypes, maintaining the selection to favor complete segregation of mutant cyanobacterial chromosomes.
- Grow mutant clones in 100ml of BG-11.