The previous attempt at sonication (see 20100618) failed, likely due to no using the correct equipment (tubes and Covaris adapter). The two gDNA pools, which had previously been unsuccessfully fragmented on 20100618 (SB and WB) were sonicated using a Covaris S2. Used the guidelines of the manufacturer (listed below) for shearing gDNA to a desired target size (500bp):

Duty Cycle: 5%

Intensity: 3

Cycels per Burst: 200

Time (seconds): 90

Temp (water bath): 4C

Power Mode: Frequency Sweeping

Sample Volume: 120uL

Buffer: TE

DNA Mass: ~8ug

Starting Material: >50kb

AFA Intensifier tubes and associated Covaris adapter.

After shearing, ran 250ng of each pool on a 2% TAE agarose gel for fragmentation verification.

Results:

Lane 1 – Hyperladder I

Lane 2 – R37

Lane 3 – R51

Looking at this gel, the samples have been successfully fragmented and I would estimate have generated and average fragment size of ~400bp (going from bottom to top of the Hyperladder: 200bp, 400bp, 600bp, 800bp, 1000bp). So, this looks great! Can proceed with remainder of MeDIP procedure at any time.

Additionally, I will confirm a more accurate assessment of average fragment size by running these two samples on the Agilent Bioanalyzer.