840:153g:Projects/project23/2012/10/18 - Revision history

Casey Durnan: /* PCR Amplification of g. sulfereducens DNA */

2012-10-19T00:10:31Z

PCR Amplification of g. sulfereducens DNA

←Older revision		Revision as of 00:10, 19 October 2012
Line 21:		Line 21:
	** 11 - Downstream fragment at 65°C		** 11 - Downstream fragment at 65°C

-	[[Image:PCR_gel.jpg]]	+	[[Image:PCR_gel.jpg\|600px]]

	*Conclusions:		*Conclusions:

Casey Durnan: /* PCR Amplification of g. sulfereducens DNA */

2012-10-18T23:34:33Z

PCR Amplification of g. sulfereducens DNA

←Older revision		Revision as of 23:34, 18 October 2012
Line 7:		Line 7:
	<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
	==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]]
		+
		+	*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.
		+
		+



	<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
-	\|}	+

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

Casey Durnan: /* PCR Amplification of g. sulfereducens DNA */

2012-10-18T20:20:03Z

PCR Amplification of g. sulfereducens DNA

←Older revision		Revision as of 20:20, 18 October 2012
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
		+

Casey Durnan at 20:11, 18 October 2012

2012-10-18T20:11:15Z

←Older revision		Revision as of 20:11, 18 October 2012
Line 1:		Line 1:
	{\|{{table}} width="800"		{\|{{table}} width="800"
	\|-		\|-
-	\|style="background-color: #EEE"\|[[Image:owwnotebook_icon.png\|128px]]<span style="font-size:22px;"> ~~Project name~~</span>	+	\|style="background-color: #EEE"\|[[Image:owwnotebook_icon.png\|128px]]<span style="font-size:22px;"> Cloning of the omcf gene</span>
	\|style="background-color: #F2F2F2" align="center"\|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}\|0\|-11}}\|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}\|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>      </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}\|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}		\|style="background-color: #F2F2F2" align="center"\|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}\|0\|-11}}\|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}\|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>      </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}\|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
	\|-		\|-
	\| colspan="2"\|		\| colspan="2"\|
	<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->		<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
-	==~~Entry title~~==	+	==PCR Amplification of g. sulfereducens DNA==
-	* ~~Insert content here~~...	+	* 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.

Casey Durnan: Autocreate 2012/10/18 Entry for 840:153g:Projects/project23

2012-10-18T19:56:40Z

Autocreate 2012/10/18 Entry for 840:153g:Projects/project23

New page

{|{{table}} width="800"
|-
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>      </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|-
| colspan="2"|

==Entry title==
* Insert content here...


|}

__NOTOC__
[[category:OWWLabNotebookV1]]