Hi all!
Shearing gDNA for NGS sample prep. I'm trying to shear my DNA into 500bp fragments using the bioruptor and have two problems:
Most importantly:
Input = 5ug of DNA in 100ul 1xTE in a LoBind 1.5ml tube
= 50ng/ul on nanodrop
Output = 50ng/ul on nanodrop, but only ~25ng/ul on bioanalyser! Does this mean only half of my DNA is sheared/detectable on DNA 1000 chip? How can I make it shear more of my sample?
Secondly, I'm having trouble getting a tight size range - the settings on the website (6x 90s OFF - 15s ON High Intensity) gave me fragments of around 1500bp. After some fiddling I got an average of 534bp with 10mins 30s OFF 30s ON High Intensity. Size range is still quite broad though, any tips on narrowing it?
HUGE appreciation for any help
Shearing gDNA for NGS sample prep. I'm trying to shear my DNA into 500bp fragments using the bioruptor and have two problems:
Most importantly:
Input = 5ug of DNA in 100ul 1xTE in a LoBind 1.5ml tube
= 50ng/ul on nanodrop
Output = 50ng/ul on nanodrop, but only ~25ng/ul on bioanalyser! Does this mean only half of my DNA is sheared/detectable on DNA 1000 chip? How can I make it shear more of my sample?
Secondly, I'm having trouble getting a tight size range - the settings on the website (6x 90s OFF - 15s ON High Intensity) gave me fragments of around 1500bp. After some fiddling I got an average of 534bp with 10mins 30s OFF 30s ON High Intensity. Size range is still quite broad though, any tips on narrowing it?
HUGE appreciation for any help
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