Tag Archives: Covaris S2

gDNA Sonication – SB/WB gDNA pools (prep for MeDIP) from 20100618

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.

gDNA Sonication – SB/WB gDNA pools (prep for MeDIP) from earlier today

The two gDNA pools (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

To be noted, the Covaris guidelines list the use of an “AFA Intensifier” tube, which I did not use (because we don’t have them).

After shearing, ran 250ng of each pool on a 2% TAE agarose gel for fragmentation verification. Also ran 250ng of pre-sonication DNA from each pool as controls.

Results:

Lane 1 – Hyperladder I

Lane 2 – R37, Un-sonicated

Lane 3 – R37, sonicated

Lane 4 – R51, Un-sonicated

Lane 5 – R51, sonicated

Sonication with the Covaris did NOT produce the desired fragmentation (500bp smear) in either sample, although the R37 sonicated samples shows a significantly greater degree of fragmentation than the R51 sonicated sample. Not sure how to explain this difference, other than the R51 sample has a greater amount of DNA. Additionally, the results could be explained by the fact that we did not use the AFA Intensifier listed in the Covaris guidelines…

Am consulting with a person in Genome Sciences who has used a Covaris for DNA fragmentation in the past to see if the AFA Intensifiers are indeed necessary and, if so, we can use two of them. Hopefully have an answer soon and be able to proceed with additional fragmentation next week.