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Project:Sex typing with amelogenin/results-20120301: Difference between revisions
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Project:Sex typing with amelogenin/results-20120301 (view source)
Revision as of 21:32, 2 March 2012
, 2 March 2012Added a bit about oils
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== Experiment 1 == | == Experiment 1 == | ||
Visualised the previous night's PCR run on 1% agarose gel with 10ul/100ml ethidium bromide stock solution. This is twice the amount of EtBr as usual, but it doesn't make a difference (see below). | Visualised the previous night's PCR run on 1% agarose gel with 10ul/100ml ethidium bromide stock solution. This is twice the amount of EtBr as usual, but it doesn't make a difference (see below). | ||
We did not cover the master mix / primers / template solution with oil. The PCR reaction vessels had condensed liquid all over the vessel, including up near the lid. I (Nicholas) theorise that the mixture evaporated, inhibiting PCR, in some cases severely. | |||
[[File:2012-02-29-results-1.JPG|200px|thumb|none|Results of experiment 1 (Empty lane, ladder, Nicholas, Helena, Mike, Tonderai)]] | [[File:2012-02-29-results-1.JPG|200px|thumb|none|Results of experiment 1 (Empty lane, ladder, Nicholas, Helena, Mike, Tonderai)]] | ||
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* Ensure the alcohol was mixed with the tenderiser solution, in order to precipitate out the most DNA | * Ensure the alcohol was mixed with the tenderiser solution, in order to precipitate out the most DNA | ||
* Ensure that the DNA, when extracted from the alcohol / saliva / tenderiser solution (yuck), was well dried, by heating for 5 min at 55 degrees. | * Ensure that the DNA, when extracted from the alcohol / saliva / tenderiser solution (yuck), was well dried, by heating for 5 min at 55 degrees. | ||
I tried two different oils: | |||
* "Laser Lube" (Thanks to Hipster for this description) -- machine lubricant, from a can near the drill press | |||
* A mysterious bottle of oil I found in the biohacking box, which could be mineral oil (though it's not the same bottle that we were using before). | |||
I used the old master mix, as we have run out of the new stuff. | I used the old master mix, as we have run out of the new stuff. | ||
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Visualised on 1% agarose gel with 6ul/100ml ethidium bromide solution. NB we have previously been using 5ul/100ml. Based on these results I am not convinced that more EtBr is at all helpful -- I noticed no difference in brightness between these two runs. | Visualised on 1% agarose gel with 6ul/100ml ethidium bromide solution. NB we have previously been using 5ul/100ml. Based on these results I am not convinced that more EtBr is at all helpful -- I noticed no difference in brightness between these two runs. | ||
[[File:2012-03-01-results-1.JPG|200px|thumb|none|Results of experiment 1 (Empty lane, ladder, Nicholas - meat tenderiser, Nicholas - meat tenderiser, Nicholas - heat to lyse)]] | [[File:2012-03-01-results-1.JPG|200px|thumb|none|Results of experiment 1 (Empty lane, ladder, Nicholas - meat tenderiser and laser lube, Nicholas - meat tenderiser and mystery oil, Nicholas - heat to lyse and mystery oil)]] | ||
Once again, two distinct bands are visible, but they don't seem to be in the right places on the gel. | Once again, two distinct bands are visible, but they don't seem to be in the right places on the gel. | ||
I conclude that we can use either mystery oil or laser lube. | |||
This is looking much more promising. It seems that it will now be a matter of fine tuning. | This is looking much more promising. It seems that it will now be a matter of fine tuning. | ||
Also, the orange filter is amazing! Thanks to Stu. | Also, the orange filter is amazing! Thanks to Stu. | ||
