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DNA extraction from whole blood

blood DNA extraction

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6 replies to this topic

#1 moerae

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Posted 26 May 2013 - 08:04 PM

I'm putting this up for a colleague of mine.

We use the Gentra Puregene kit to extract DNA from whole blood. The protocol lyse the RBC first before lysing the WBC with their respective lysing solution at certain ratios. We had an incident the other day whereby we've added the wrong ratio of RBC lysis buffer for one sample (too much), and the wrong ratio of WBC lysis buffer for another sample (too much). We carried on with the protocol to see what would happen, and the end result is very little DNA being extracted.

The question we have is why.... if we're adding too much lysis buffer would it not just lyse all the cells anyway and not matter?

#2 Curtis

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Posted 27 May 2013 - 12:16 AM

Did you realize you might have diluted the extracted DNA by adding too much lysis buffer?

#3 moerae

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Posted 27 May 2013 - 01:24 AM

We always pellet it down after the addition of each lysis buffer so I'd assume that we wouldn't have diluted out our DNA. Well at least that's what I thought would happen.

#4 almost a doctor

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Posted 27 May 2013 - 01:47 AM

Did you adjust the volumes of the other buffers (post-lysis) for the samples that you added "too much" lysis buffer?

I don't think diluting DNA is the problem, but diluting the other buffers. Unless you added "too much" of the other buffers (to compensate for the "too much" RBC and WBC lysis buffers) so that concentrations remain the same, by having higher volumes post-lysis your precipitating salts will be diluted which will result in poorer DNA extraction.

Hope this makes sense, somehow I feel I'm not explaining myself too well Posted Image

#5 moerae

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Posted 27 May 2013 - 12:16 PM

Yeah, we adjusted the volumes once we realised it was too much to go back to what it "should" be. Would that dilute out the other buffers?

#6 almost a doctor

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Posted 28 May 2013 - 12:55 AM

Yeah, we adjusted the volumes once we realised it was too much to go back to what it "should" be. Would that dilute out the other buffers?


Which volumes did you adjust to "go back to what it should be"?

What I meant in my post is that you should have added more of the subsequent buffers to match the "too much" lysis buffer, so that the concentration of reagents remained the same. If you reduced the lysed cells volume back to "what it should be", you've reduced your input sample and therefore reduce your DNA yield. If this is not what you did, then forget what I've just said Posted Image

#7 moerae

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Posted 28 May 2013 - 05:33 PM

Hi,

Thanks for that. I think that's where we went wrong. The input for the DNA ppt step was the problem. Thank you





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