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Old 10-10-2012, 02:09 AM   #1
addyblanch
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Location: Nottingham

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Unhappy High concentration Covaris Shearing

Hi all,

I'm currently using the Covaris S2 to fragment my DNA to approximately 1.5-2kb.
Mainly so I can ligate to perform an inverse PCR before NGS library prep. My problem seems to be when I need to analyse the fragment pattern on a gel. As I only have 130ul I struggle to add enough to visualise using ethidium bromide.
is it possible to fragment a higher concentration, i've read its independent but wondered if sonicating for a longer time would have a detrimental effect on my DNA.

Are there any other ways round this??


Many thanks
Adam
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Old 10-10-2012, 07:17 AM   #2
pmiguel
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Hi Adam,

I can think of a couple of ways:

(1) Use a more sensitive stain for your agarose gel. Eg, SYBR-gold.
(2) Run a sheared DNA aliquot on an Agilent High Sensitivity chip -- but you would probably need to dilute it at least 1:5 to prevent massively overloading it. Also, you only get a picture below 10 kb or so. If you happen to have left some of the genomic DNA un-sheared, you would not see it. (But the person with their sample in the next well might...
(3) Take the 30 ul and do a quick butanol extraction on it to reduce its volume down to something you can load in on your agarose EtBr gel.

--
Phillip
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Old 10-11-2012, 02:36 AM   #3
addyblanch
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Thats great thanks!
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