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DNA extraction problem - strange bands

DNA extraction

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#1 ginja1980

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Posted 12 November 2013 - 01:49 PM

Hello,

 

I am working on some DNA extractions and getting some very odd patterns. I am attaching a picture of a gel.

 

First we carried out a phenol-chloroform extraction. We detected some impurities, so ran a Qiagen DNAeasy clean up on these extractions. This caused some degradation. This is all expected. Some of the P-C only samples were degraded as well, but the extra step caused more degradation. 

 

What is bothering me is the banding pattern observed at low molecular weights. It is more visible in the more degraded samples. Has anybody seen anything like this before? 

 

We have checked for contamination. These are RNase treated. The most stumping thing is the pattern is always stronger in the degraded samples. We have checked the loading buffers. Run the gels with EtBr and gel red. Is the genomic DNA fragmenting in a regular fashion (that sounds crazy).

 

In the gel pictures, extractions from the same individual are side by side and have the same letter, the number denotes how many extraction steps were taken (1= P-C only, 2= P-C & DNAeasy)

 

Any ideas, or thoughts are most welcome. Everyone around here is a bit stumped. 

 

This DNA is from various species of bee (Bombus sp.)

 

Thank you in advance for any help you can provide!

 

 

Attached Thumbnails

  • 20131111_gelpic.jpg


#2 Celz

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Posted 12 November 2013 - 04:17 PM

Those low MW bands are the degraded DNA. 



#3 ginja1980

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Posted 13 November 2013 - 01:31 AM

Thank you for the response. I was aware that it was degraded DNA, but it was the banding pattern that was confusing me, as degraded DNA just looks like a smear. I having been asking around, it looks very similar to DNA from apoptotic cells. So it looks like it might be related to that mechanisms (eg breaks in the DNA are being made in a similar fashion)

 

http://www.sciencedi...03269798929293#

 

Ian



#4 mushroom911

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Posted 05 December 2013 - 08:31 PM

I had similar experience using Qiagen dna extraction kit. I can't identify the cause for the band as well.wacko.png After struggling for a period of time, i gave up. I juz ignore the band carry on downstream analysis. Luckily, the experiment went well. tongue.png I suggest you to carry on if your experiment don't require high quality dna sample. If it does, maybe you should redo the dna extraction.

 

All the best in your study.



#5 phage434

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Posted 06 December 2013 - 08:07 PM

I've seen RNA contamination of these samples. Are the bands at 1500 and 2700 bp?  I don't think these kits have RNAse in the resuspension buffer.







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