Go for sequencing although big peaks?

frensch

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Join Date: May 2013

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Go for sequencing although big peaks?

07-28-2013, 01:35 AM

Hi,
I am working with mouse heart homogenates.
Some points: mince heart with razor blades, 5min fix, 60*up and down with dounce homogenizer, 12Cycles (Bioprupter Diagenode).
With the named conditions I did a gel with Agilent and I couldn´t see a peak in my Chiped DNA. Is this normal? My DNA concentration is 0.1ng/µl, volume 44µl. I could see a really nice peak in my Inputsamples at 200bp. BUT I also see a peak at ~2kb. The ration between the small desirable peak and the big peaks are 2/3 small peak and 1/3 big peaks.
I changed different conditions, 10 and 15 min fix, 30*dounce, 5 to 20 Cycles; but it doesn´t look good in the Agilent gel. With these conditions I also get bands with 2kbs.
I have heard that many people had these problems to get big peaks...
Regardless these big peaks, do you think I can go for sequencing with these samples?
Attached Files

Agilent results.pdf (136.9 KB, 152 views)
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