Ladder-like bioanalyzer trace on supposed 150bp DNA sample library

Ace5858 · 03-31-2012, 01:56 PM

I recently started working on very old hyde tissue, extracting DNA, running a HS Bioanalyzer chip. My results showed that the samples were heavily degraded down to 30bp to 50bp fragments. When I made the libraries, theoretically I should see 130bp to 150bp fragments with the adapters attached. I did a PCR amplification to enrich the libraries. I didn't size select because there was no larger pieces than 50bp in the original bioA run.

However, when I ran the second HS bioA chip with these libraries, I got this strange ladder-like read on my samples with extremely high basepairs.

Has anyone else experienced this same strange result?

Lampshade_Page_12.jpg

pmiguel · 03-31-2012, 02:39 PM

First, you bioA chip results are hosed in some of the lanes. You can see the fuzzy 10.38kb upper marker has not been correctly identified in several of the lanes. You need to view the .xad file with 2100 Expert software and go to each of those traces, switch to the peak table tab and figure out which peak is your real 10.38 kb band, right-click on it and choose "manually set upper marker". This will give you the correct size of your peaks.

That said, looks to me like the right four lanes have 2 different MW markers in them. Either you samples are contaminated with them, or the pins of your bioA were contaminated with them.

That is my take, anyway.
--
Phillip

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03-31-2012, 01:56 PM	#1
Ace5858 Junior Member Location: New York Join Date: Mar 2012 Posts: 8	Ladder-like bioanalyzer trace on supposed 150bp DNA sample library I recently started working on very old hyde tissue, extracting DNA, running a HS Bioanalyzer chip. My results showed that the samples were heavily degraded down to 30bp to 50bp fragments. When I made the libraries, theoretically I should see 130bp to 150bp fragments with the adapters attached. I did a PCR amplification to enrich the libraries. I didn't size select because there was no larger pieces than 50bp in the original bioA run. However, when I ran the second HS bioA chip with these libraries, I got this strange ladder-like read on my samples with extremely high basepairs. Has anyone else experienced this same strange result? Lampshade_Page_12.jpg

03-31-2012, 02:39 PM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	First, you bioA chip results are hosed in some of the lanes. You can see the fuzzy 10.38kb upper marker has not been correctly identified in several of the lanes. You need to view the .xad file with 2100 Expert software and go to each of those traces, switch to the peak table tab and figure out which peak is your real 10.38 kb band, right-click on it and choose "manually set upper marker". This will give you the correct size of your peaks. That said, looks to me like the right four lanes have 2 different MW markers in them. Either you samples are contaminated with them, or the pins of your bioA were contaminated with them. That is my take, anyway. -- Phillip