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Old 03-03-2015, 12:49 PM   #6
Myrmex
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Location: Chicago, IL

Join Date: Dec 2014
Posts: 9
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Quote:
Originally Posted by nucacidhunter View Post
I can think of three reasons:

1- Pipetting inaccuracy caused by calibration/maintenance issues or operator
2- Presence of viscous material in DNA preps (mostly in non-column based extractions). This would show as streaks in wells of gel. This can be avoided by spinning DNA tube or plate at high speed for 5 min to precipitate non-soluble materials and transferring from top of wells.
3- Proper mixing of DNA with reagents or after dilution
Okay, thank you, this makes a lot of sense. We do have viscous material in our CTAB extractions which is one of the ideas we came up with for why this is happening. When I decrease the volume of the sample in a SpeedVac from 100Ál to 50Ál the material gets a lot more viscous. I have been trying to mix things very well before measuring on the Qbit but now I realize that maybe I should be spinning them down first and taking only the water liquid on the top.

The other option I guess is to re-extract with a column extraction- I was just worried about getting enough DNA, as we are already riding a thin line far as that is concerned...
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