840:153g:Projects/project23/2012/10/25: Difference between revisions
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==Continuation of the Mutagenesis Reaction == | ==Continuation of the Mutagenesis Reaction == | ||
* On Tuesday, we | *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 | ||
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*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 | ||
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* | *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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__NOTOC__ | __NOTOC__ | ||
[[category:OWWLabNotebookV1]] | [[category:OWWLabNotebookV1]] |
Revision as of 15:51, 29 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> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
Continuation of the Mutagenesis Reaction
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