840:153g:Projects/project23/2012/10/18: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
Line 8: Line 8:
==PCR Amplification of g. sulfereducens DNA==
==PCR Amplification of g. sulfereducens DNA==
* On Tuesday, we amplified or gene of interest from geobacter sulfereducens DNA.  Since we need to remove an internal restriction site in our gene, we preformed a point mutation.  This was done by amplifying two reactions.  This creates two sections with an small overlapping sequence over the mutation site.  Our oligos contain a base-change so that an overwhelming majority of the PCR product should not have the restriction site any longer.  We also ran each reaction at three different annealing temperatures since our particular oligos are potentially problematic.   
* On Tuesday, we amplified or gene of interest from geobacter sulfereducens DNA.  Since we need to remove an internal restriction site in our gene, we preformed a point mutation.  This was done by amplifying two reactions.  This creates two sections with an small overlapping sequence over the mutation site.  Our oligos contain a base-change so that an overwhelming majority of the PCR product should not have the restriction site any longer.  We also ran each reaction at three different annealing temperatures since our particular oligos are potentially problematic.   
* On Thursday, we ran our reactions and controls from Tuesday on a 1% agarose gel to check for intended PCR product.
* On Thursday, we ran our reactions and controls from Tuesday on a 1% agarose gel to check for intended PCR product. We loaded our samples as follows:
** 1 -  Upstream fragment at 55°C
** 2 -  Upstream fragment at 60°C
** 3 -  Upstream fragment at 65°C
** 4 -  100 bp ladder
** 5 -  Upstream negative control
** 6 -  Positive control
** 7 -  Downstream negative control
** 8 -  100 bp ladder
** 9 -  Downstream fragment at 55°C
** 10 - Downstream fragment at 60°C
** 11 - Downstream fragment at 65°C
 




Revision as of 13:20, 18 October 2012

Cloning of the omcf gene <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

PCR Amplification of g. sulfereducens DNA

  • On Tuesday, we amplified or gene of interest from geobacter sulfereducens DNA. Since we need to remove an internal restriction site in our gene, we preformed a point mutation. This was done by amplifying two reactions. This creates two sections with an small overlapping sequence over the mutation site. Our oligos contain a base-change so that an overwhelming majority of the PCR product should not have the restriction site any longer. We also ran each reaction at three different annealing temperatures since our particular oligos are potentially problematic.
  • On Thursday, we ran our reactions and controls from Tuesday on a 1% agarose gel to check for intended PCR product. We loaded our samples as follows:
    • 1 - Upstream fragment at 55°C
    • 2 - Upstream fragment at 60°C
    • 3 - Upstream fragment at 65°C
    • 4 - 100 bp ladder
    • 5 - Upstream negative control
    • 6 - Positive control
    • 7 - Downstream negative control
    • 8 - 100 bp ladder
    • 9 - Downstream fragment at 55°C
    • 10 - Downstream fragment at 60°C
    • 11 - Downstream fragment at 65°C


Retrieved from "https://openwetware.org/mediawiki/index.php?title=840:153g:Projects/project23/2012/10/18&oldid=636590"