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DNA extraction problems.

Tick. DNA

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#1 Bioscientist

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Posted 15 August 2014 - 02:17 PM

Hello

 

I have a protocol for extracting DNA from ticks. The ticks that I am using are preserved in glycerol.

 

I wash them with 70% ethanol and then sterile water and then extract their DNA. I used 1 tick from the same vial each time.

 

The problem is at the end (after ethanol precipitation)  I sometimes see the DNA pellet and sometimes I don't. Like I tried it 10 times. And I failed to get a DNA pellet 5 times. Any idea why this can happen? Is it that particular tick sample? Sometimes the tick have a missing leg, does that cause nuclease to get out and I don't get DNA?

 

I looking forward to your suggestions.

 

Thanks!

 

Troubled



#2 bob1

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Posted 15 August 2014 - 02:44 PM

There are a few scenarios where not seeing a pellet is likely - one of the more common is that the pellet was there, but has been washed off the tube.  Another reason might be that the pellet isn't visible, they can be quite faint, but there is still DNA there. The best thing you can do, is just proceed with the extraction, and then dissolve the "pellet" as you would normally, then check for DNA presence on a spec and on a gel.

 

If you are worried about yield, you can use a carrier such as linear polyacryamide to help increase the yield.



#3 phage434

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Posted 15 August 2014 - 05:31 PM

Also, dyed glycogen (Novagen pellet-paint e.g.) will co-precipitate and make the pellet visible.



#4 hobglobin

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Posted 17 August 2014 - 04:25 AM

Apart from the above suggestions, which you should follow, you might have problems to get high quality DNA, because glycerol isn't really a good  agent to preserve samples...hopefully the ticks were cooled or frozen and stored only short time.

Therefore even a nice pellet might not help you if the DNA is too degraded, and only a gel (and/or PCR) will give you information you about the quality and usability.


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