User:Stuart McKellar/Notebook/Bird Sex Testing/2012/11/14

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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.

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