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.