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Forum: Epigenetics 10-07-2014, 09:04 PM
Replies: 0
Views: 1,557
Posted By nfourier
genomic DNA shearing with Covaris 12*12 tubes

Hello, I am calibrating a new protocol and I need to shear my cross-linked DNA (2*10^7 RAW cell line) to an average size of 1000 bps. I'm using Covaris E220 with12*12 tubes. I did a time course...
Forum: Epigenetics 01-06-2013, 03:50 AM
Replies: 16
Views: 8,122
Posted By nfourier
Fixation protocol

Thank you Hamid.
I will take your advice and use Ethan's protocol. I prepared the buffers but I have a question regarding cells in suspention. Ethan's protocol has the fixation buffer volumes for...
Forum: Epigenetics 12-23-2012, 06:27 AM
Replies: 16
Views: 8,122
Posted By nfourier
Hello Hamid, 1) I fix my cells in their medium,...

Hello Hamid,
1) I fix my cells in their medium, in formaldehyde (1.42% final concentration) for 15min in room temperature. It had worker before with 3 different cell lines in our lab.
2) I prepare...
Forum: Epigenetics 12-20-2012, 03:22 AM
Replies: 16
Views: 8,122
Posted By nfourier
I did another run of sonications. I used Covaris...

I did another run of sonications. I used Covaris shearing buffer (as suggested by Hamid) and tried 3 setups (each sample is 130ul, ~2.5*10^6 cells):
duty cycle 2%, 8min
duty cycle 5%, 8min
duty...
Forum: Epigenetics 12-18-2012, 11:11 PM
Replies: 16
Views: 8,122
Posted By nfourier
Thank you Hamid. I will try the shearing buffer....

Thank you Hamid. I will try the shearing buffer. I saw in the gel that the lines are 2-10min, what does that mean? what is the exact time? and what is the duty cycle?
Forum: Epigenetics 12-18-2012, 06:23 AM
Replies: 16
Views: 8,122
Posted By nfourier
I read in the Covaris optimization protocol to do...

I read in the Covaris optimization protocol to do a time course of 2-12min and do 6 time points. This is why making changes in time duration of 5-10s seems puzzling to me...
Forum: Epigenetics 12-18-2012, 04:11 AM
Replies: 16
Views: 8,122
Posted By nfourier
Thank you. How come the treatment time is 50/80...

Thank you.
How come the treatment time is 50/80 sec? In the time course Covaris recommends its 2-12 min...
Forum: Epigenetics 12-18-2012, 02:01 AM
Replies: 16
Views: 8,122
Posted By nfourier
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...
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