840:153g:Projects/project23/2012/10/25 - Revision history

Casey Durnan: /* Continuation of the Mutagenesis Reaction */

Casey Durnan — Tue, 30 Oct 2012 00:29:41 GMT

Continuation of the Mutagenesis Reaction

←Older revision		Revision as of 00:29, 30 October 2012
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-	*After analyzing this gel, we decided to run another PCR to do a side-by-side comparison of the two fragments and the combined reaction, to see if they are separate products. We used only the 1:200 template, and the 65°C annealing temperature, as they gave us the cleanest images to date. We prepared an upstream and downstream reaction from our original total-DNA sample, and primered/ non-primered set identical to earlier this week. We also prepared each of these sets without template DNA for negative control. We cycled our PCR with the same protocol we used in the past, but with a non-gradient annealing temperature.	+	*After analyzing this gel, we decided to run another PCR to do a side-by-side comparison of the two fragments and the combined reaction, to see if they are separate products. We used only the 1:200 template, and the 65°C annealing temperature, as they gave us the cleanest images to date. We prepared an upstream and downstream reaction from our original total-DNA sample, and primered/ non-primered set identical to earlier this week. We also prepared each of these sets without template DNA for negative control. Lastly, we prepared 6 more primered samples to extract DNA from if we prove to have our intended product. We cycled our PCR with the same protocol we used in the past, but with a non-gradient annealing temperature.

Casey Durnan: /* Continuation of the Mutagenesis Reaction */

Casey Durnan — Tue, 30 Oct 2012 00:20:55 GMT

Continuation of the Mutagenesis Reaction

←Older revision		Revision as of 00:20, 30 October 2012
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	**Template dilution works well for either high or low concentrations. We will use 1:200 for further amplification.		**Template dilution works well for either high or low concentrations. We will use 1:200 for further amplification.
	**Annealing temperature did not seem to vary results for this range.		**Annealing temperature did not seem to vary results for this range.
		+
		+
		+	*After analyzing this gel, we decided to run another PCR to do a side-by-side comparison of the two fragments and the combined reaction, to see if they are separate products. We used only the 1:200 template, and the 65°C annealing temperature, as they gave us the cleanest images to date. We prepared an upstream and downstream reaction from our original total-DNA sample, and primered/ non-primered set identical to earlier this week. We also prepared each of these sets without template DNA for negative control. We cycled our PCR with the same protocol we used in the past, but with a non-gradient annealing temperature.

Casey Durnan: /* Continuation of the Mutagenesis Reaction */

Casey Durnan — Mon, 29 Oct 2012 22:53:01 GMT

Continuation of the Mutagenesis Reaction

←Older revision		Revision as of 22:53, 29 October 2012
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	==Continuation of the Mutagenesis Reaction ==		==Continuation of the Mutagenesis Reaction ==

-	*On Tuesday, we prepared to combine our upstream and downstream fragments in the final mutagenesis step. First a high concentration of 1:20 template DNA, and a low concentration of 1:200 were prepared with water for each the upstream and downstream fragments. We then prepared our samples to amplify. Our approach was to mix the fragments together with ~~and~~ without primers to try to identify an effective combination to create our entire gene. For each set we also investigated the possible effect of template concentration and annealing temperature, for a total of 12 different combinations. Our nomenclature used is three-part, and outlined below.	+	*On Tuesday, we prepared to combine our upstream and downstream fragments in the final mutagenesis step. First a high concentration of 1:20 template DNA, and a low concentration of 1:200 were prepared with water for each the upstream and downstream fragments. We then prepared our samples to amplify. Our approach was to mix the fragments together with or without primers to try to identify an effective combination to create our entire gene. For each set we also investigated the possible effect of template concentration and annealing temperature, for a total of 12 different combinations. Our nomenclature used is three-part, and outlined below.
		+
		+

	*1.)Primer configuration		*1.)Primer configuration
Line 53:		Line 55:


-	*We ran these on a 1% agarose gel for 30min at 120V	+	*We ran these samples on a 1% agarose gel for 30min at 120V

Casey Durnan: /* Continuation of the Mutagenesis Reaction */

Casey Durnan — Mon, 29 Oct 2012 22:51:12 GMT

Continuation of the Mutagenesis Reaction

←Older revision		Revision as of 22:51, 29 October 2012
Line 8:		Line 8:
	==Continuation of the Mutagenesis Reaction ==		==Continuation of the Mutagenesis Reaction ==

-	* On Tuesday, we ~~ran~~ the ~~gel for last week's PCR products~~. ~~The~~ high concentration of 1:20 template DNA, and a low concentration of 1:200 were prepared with water for each the upstream and downstream fragments. Our nomenclature used is three-part, and outlined below.	+	*On Tuesday, we prepared to combine our upstream and downstream fragments in the final mutagenesis step. First a high concentration of 1:20 template DNA, and a low concentration of 1:200 were prepared with water for each the upstream and downstream fragments. We then prepared our samples to amplify. Our approach was to mix the fragments together with and without primers to try to identify an effective combination to create our entire gene. For each set we also investigated the possible effect of template concentration and annealing temperature, for a total of 12 different combinations. Our nomenclature used is three-part, and outlined below.

	*1.)Primer configuration		*1.)Primer configuration
Line 18:		Line 18:
	*3.)Annealing temperature for PCR, in °C		*3.)Annealing temperature for PCR, in °C

		+	*We used the same PCR protocol as last week.
		+
		+
		+	*On Thursday, we ran the gel for Tuesday's PCR products.

	*Our positive control was standard template DNA and associated primers from lab stock		*Our positive control was standard template DNA and associated primers from lab stock
Line 37:		Line 41:


-	*~~Bottom~~ Lane:	+	*Lower Lane:
	#100 bp ladder		#100 bp ladder
	#N+55		#N+55
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	*Results:		*Results:
		+
		+
		+	*Upper Lane
		+	[[Image:10.25.12_Primered_Lanes.jpg\|600px]]
		+
		+
		+	*Lower Lane
		+	[[Image:10.25.12_Non-primered_Lanes.jpg\|600px]]
		+
		+
		+	*Conclusions:
		+	**Our positive control checked ok, indicating working PCR reagents
		+	**Our negative control showed a faint band in the <100bp region, likely due to primers. Nothing else appeared, indicating an uncontaminated working stock.
		+	**Our non-primered set did not work. This should, in theory, so perhaps we need to adjust the thermocycle protocol.
		+	**Our primered set did amplify a numer of distinct bands. Our target length of ~400bp showed bands on both the upper and lower boundaries. One of these may be our intended product, as there is some range of error in marker ladders. Perhaps none of the bands are our product, however, and are just PCR artifacts.
		+	**Template dilution works well for either high or low concentrations. We will use 1:200 for further amplification.
		+	**Annealing temperature did not seem to vary results for this range.


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Casey Durnan: /* Continuation of the Mutagenesis Reaction */

Casey Durnan — Mon, 29 Oct 2012 22:14:47 GMT

Continuation of the Mutagenesis Reaction

←Older revision		Revision as of 22:14, 29 October 2012
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	==Continuation of the Mutagenesis Reaction ==		==Continuation of the Mutagenesis Reaction ==

-	* On Tuesday, we ran the gel for last week's PCR products. ~~This was done on~~ a ~~dual~~-~~lane chamber~~, ~~loaded as follows:~~	+	* On Tuesday, we ran the gel for last week's PCR products. The high concentration of 1:20 template DNA, and a low concentration of 1:200 were prepared with water for each the upstream and downstream fragments. Our nomenclature used is three-part, and outlined below.

		+	*1.)Primer configuration
		+	**N = no primers used
		+	**P = outside primers used
		+	*2.)Template DNA concentration
		+	** + = 1:20
		+	** - = 1:200
		+	*3.)Annealing temperature for PCR, in °C
		+
		+
		+	*Our positive control was standard template DNA and associated primers from lab stock
		+	*Our negative control had outside primers and no template
		+
		+
		+	*We loaded our samples as follows:

	*Top Lane:		*Top Lane:
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	#N+60		#N+60
	#N+65		#N+65
-	#~~Positive~~ Control	+	#Negative Control
	#N-55		#N-55
	#N-60		#N-60

Casey Durnan: /* Continuation of the Mutagenesis Reaction */

Casey Durnan — Mon, 29 Oct 2012 21:54:39 GMT

Continuation of the Mutagenesis Reaction

←Older revision		Revision as of 21:54, 29 October 2012
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	<!-- ##### 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. ##### -->
	==Continuation of the Mutagenesis Reaction ==		==Continuation of the Mutagenesis Reaction ==
-	*	+
		+	* On Tuesday, we ran the gel for last week's PCR products. This was done on a dual-lane chamber, loaded as follows:
		+
		+
		+	*Top Lane:
		+	#100 bp ladder
		+	#P+55
		+	#P+60
		+	#P+65
		+	#Positive Control
		+	#P-55
		+	#P-60
		+	#P-65
		+	#100 bp ladder
		+
		+
		+	*Bottom Lane:
		+	#100 bp ladder
		+	#N+55
		+	#N+60
		+	#N+65
		+	#Positive Control
		+	#N-55
		+	#N-60
		+	#N-65
		+	#100 bp ladder
		+
		+
		+	*We ran these on a 1% agarose gel for 30min at 120V
		+
		+
		+	*Results:
		+
		+

Valerie Wisco at 19:48, 25 October 2012

Valerie Wisco — Thu, 25 Oct 2012 19:48:11 GMT

←Older revision		Revision as of 19:48, 25 October 2012
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	{\|{{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~~==	+	==Continuation of the Mutagenesis Reaction ==
-	* ~~Insert content here...~~	+	*

Valerie Wisco: Autocreate 2012/10/25 Entry for 840:153g:Projects/project23

Valerie Wisco — Thu, 25 Oct 2012 19:05:05 GMT

Autocreate 2012/10/25 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...


|}

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[[category:OWWLabNotebookV1]]