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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Cloning of the OmcF gene</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"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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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 | *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 | ||
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*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 | ||
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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. 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. | |||
Latest revision as of 22:09, 26 September 2017
Cloning of the OmcF gene | Main project page Previous entry Next entry |
Continuation of the Mutagenesis Reaction
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