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

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==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 performed a point mutation.  This was done by amplifying two reactions.  This creates two sections, one upstream and one downstream of the mutation, 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:
* On Thursday, we ran our reactions and controls from Tuesday on a 1% agarose gel, 120V for 30min, to check for intended PCR products. We loaded our samples as follows:
** 1 -  Upstream fragment at 55°C
** 1 -  Upstream fragment at 55°C
** 2 -  Upstream fragment at 60°C
** 2 -  Upstream fragment at 60°C
** 3 -  Upstream fragment at 65°C
** 3 -  Upstream fragment at 65°C
** 4 -  100 bp ladder
** 4 -  100 bp ladder
** 5 -  Upstream negative control
** 5 -  Upstream negative control (no template DNA)
** 6 -  Positive control
** 6 -  Positive control  
** 7 -  Downstream negative control
** 7 -  Downstream negative control (no template DNA)
** 8 -  100 bp ladder
** 8 -  100 bp ladder
** 9 -  Downstream fragment at 55°C
** 9 -  Downstream fragment at 55°C
** 10 - Downstream fragment at 60°C
** 10 - Downstream fragment at 60°C
** 11 - Downstream fragment at 65°C
** 11 - Downstream fragment at 65°C
[[Image:PCR_gel.jpg|600px]]
*Conclusions:
** Our positive control shows amplification. Reagents and proceedure are acceptable.
** Our negative controls show no banding except at LMW, where our oligos are present. These are points to subtract from sample wells.
** 65°C annealing runs are cleaner than lower temps for both upstream and downstream sections.
** The downstream runs show amplification in the 250-300bp region, and is distinct from the negative control. This is correct for our sequence.
** The upstream runs are very bright.  This may be due to incorrect pipetting or dissolution of oligo working stocks. There is an overabundace of DNA in these wells.
** The upstream runs may have banding in our target range of 50-100bp, however the brightness of the bands make it difficult to distinguish.  We could rerun these amplifications with lower DNA concentrations and a tighter gel, but we will assume we have the correct product and proceed to the next reaction.






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[[category:OWWLabNotebookV1]]
[[category:OWWLabNotebookV1]]

Revision as of 17:10, 18 October 2012

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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 performed a point mutation. This was done by amplifying two reactions. This creates two sections, one upstream and one downstream of the mutation, 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, 120V for 30min, to check for intended PCR products. 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 (no template DNA)
    • 6 - Positive control
    • 7 - Downstream negative control (no template DNA)
    • 8 - 100 bp ladder
    • 9 - Downstream fragment at 55°C
    • 10 - Downstream fragment at 60°C
    • 11 - Downstream fragment at 65°C

  • Conclusions:
    • Our positive control shows amplification. Reagents and proceedure are acceptable.
    • Our negative controls show no banding except at LMW, where our oligos are present. These are points to subtract from sample wells.
    • 65°C annealing runs are cleaner than lower temps for both upstream and downstream sections.
    • The downstream runs show amplification in the 250-300bp region, and is distinct from the negative control. This is correct for our sequence.
    • The upstream runs are very bright. This may be due to incorrect pipetting or dissolution of oligo working stocks. There is an overabundace of DNA in these wells.
    • The upstream runs may have banding in our target range of 50-100bp, however the brightness of the bands make it difficult to distinguish. We could rerun these amplifications with lower DNA concentrations and a tighter gel, but we will assume we have the correct product and proceed to the next reaction.
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