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Old 12-18-2012, 02:01 AM   #1
nfourier
Junior Member
 
Location: Israel

Join Date: Dec 2012
Posts: 8
Default Genomic DNA shearing with Covaris E220

I am doing a ChIP experiment and doing the genomic DNA (RAW cell line, mouse monocytes) shearing using the Covaris E220. I need my DNA fragments to be between 500-1000 bps, but 200-700 bps will be ok too.
I first started doing a time-curve using the Covaris truChIP protocol:
duty cycle: 2%
peak incident power: 105 watts
cycles per burst: 200
temp: 4-8
volume: 130 ul (with covaris microtubes).
time points: 4,8,12 min.

My buffer is from the FastChIP protocol (which has been used before in our lab, and with the same type of cells and was successful), which contains: 150mM NaCl, 50mM Tris-HCl (pH 7.5), 5 mM EDTA, NP-40 (0.5%), Triton X-100 (1%). this buffer is used for lysis and shearing.

I get fragments between 1000-3000 bps.
I am totally new to Covaris and don't know what to change first in order to get smaller fragments. Should I increase the time? Or will changing the duty cycle first be more effective (and to what %)?
I could use all the help I can get.
Attached is the gel image (from left to right: 4,8,12 min. Ladder- bold from up to down: 3000,1031,500 bps).

Thank you!
Attached Images
File Type: jpg 18.11 covaris 4-8-12min ONLy.jpg (68.7 KB, 42 views)
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