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Old 06-26-2014, 11:04 PM   #1
kit.
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Location: HK

Join Date: Feb 2013
Posts: 5
Default gDNA extraction

Hi all,

I am extracting DNA from human blood cells using Qiagen Flexigene. However, at the end when I resuspend the DNA pellet using Qiagen's FG3 buffer (10mM Tris pH 8.5). The DNA seems do not dissolve well that I found the solution sticky/viscous.

And when I measure the DNA using nanodrop, it gives multiple concentrations upon several measurement. For example, in one measurement it gives 50ng/uL, in another measurement it gives 200ng/uL, several folds increase in concentration!!

Is there any suggestion for improvement and reason for this? Many thanks.

Protocol used:
Briefly, I used Qiagen Flexigene kit. cells were lysed by Qiagen protease. subsequently after lysis add isopropanol to precipitate DNA. Followed by I wash the DNA with 70% ethanol twice. Then resuspend the pellet in FG3 buffer (10mM Tris pH 8.5) after ethanol dry.
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Old 06-26-2014, 11:30 PM   #2
elenstea
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How long do you let the pellet dry? And how much buffer do you use for resuspending? I would suggest close monitoring of the pellet while drying (not enough drying and pellet will not dissolve properly due to ethanol residues, too much drying and pellet will dissolve difficulty). If you are confident that drying time is fine then I would suggest increasing the volume of resuspension. And finally since you use nanodrop for measuring, I think it is OK to "spend" some sample for measuring with serial dilutions: undiluted, 1:10, 1:100 this would give you a much better estimate
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Old 06-27-2014, 07:43 AM   #3
rnaeye
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Leave the DNA at 55C for several hours or longer. Or you can leave at 37C overnight. I would add some EDTA in it though (0.1-1mM final concentration) High molecular weight genomic DNA takes time to dissolve. Make sure you do not over-dry the pellet. Different readings by Nanodrop suggests that DNA was not dissolved properly. Also, make sure that you have enough volume to give final concentration less than 200 ng/uL. I found that it's easiest to dissolve gDNA when it's ~100 ng/uL or lower. You can also try higher pH such as pH9, but 8.5 should be sufficient. Hope this helps.
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