User:Stuart McKellar/Notebook/Bird Sex Testing/2012/11/14: Difference between revisions
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== | ==Checking DNA in samples== | ||
* | * After yesterday, when NO DNA showed up on my gel, I washed the gel under water and put it back under the TI (transilluminator). The TI showed the ladder VERY brightly and when I looked at the lanes there was a very faint smear. Before this I added DNA to 3 of the lanes and the ladder into one lane. This will have confounded the results but it looks as though being stingy with my consumables was beneficial. | ||
The aim is to see if there is still DNA in my samples, pre-PCR. The ladder was added as a positive control to check if the gel was still able to stain the DNA. | |||
ran gel @ 300V, 60mA. No melt. Both DNA ladders were visible. The lanes contained 5ul sample and 1ul loading buffer. No visible DNA in the lanes besides the ladder. I modified the TI back to a standalone form. There should be a ton of DNA in the PCR samples too. There may not be enough contrast to see the PCR samples so I will try it in a dark room, wash the gel, and also will try a post stain maybe. This is hard to do as i don't have any GelGreen left. | |||
Checked the gel in the dark and it showed faint bands in almost all the lanes. One band had a very faint single band. Others showed many bands, suggesting non-specific binding of primers. AMG suggested raising the temperature of the annealing step as this will reduce non-specific binding. She also advised that there could be contamination. The faint bands could also be due to non-specific binding. Steps to take from here: | |||
*Try at either suggested temperature on paper, or on primers (whichever is higher), then try with the other temperature | |||
*Try 1ul of the above steps, and 2ul of the above steps. | |||
*Try using more Taq, and double check all the concentrations. | |||
Revision as of 22:05, 13 November 2012
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Checking DNA in samples
The aim is to see if there is still DNA in my samples, pre-PCR. The ladder was added as a positive control to check if the gel was still able to stain the DNA. ran gel @ 300V, 60mA. No melt. Both DNA ladders were visible. The lanes contained 5ul sample and 1ul loading buffer. No visible DNA in the lanes besides the ladder. I modified the TI back to a standalone form. There should be a ton of DNA in the PCR samples too. There may not be enough contrast to see the PCR samples so I will try it in a dark room, wash the gel, and also will try a post stain maybe. This is hard to do as i don't have any GelGreen left. Checked the gel in the dark and it showed faint bands in almost all the lanes. One band had a very faint single band. Others showed many bands, suggesting non-specific binding of primers. AMG suggested raising the temperature of the annealing step as this will reduce non-specific binding. She also advised that there could be contamination. The faint bands could also be due to non-specific binding. Steps to take from here:
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