After shearing all of the M.magister gill gDNA on 20201026, there were still three samples that still had average fragment lengths that were a bit longer than desired (~750bp, but want ~250 - 550bp):
CH05-01
CH05-21
CH07-11
I initially chose to run them on the sonicator (Bioruptor 300; Diagenode) for two successive rounds of 5 cycles (30s ON, 30s OFF; low intensity). This proved to be insufficient (see RESULTS section below), so I ran three additional successive rounds of 5 cycles (30s ON, 30s OFF; low intensity).
The original samples used 1ug of DNA in a volume of 51uL (this volume was selected to simplify downstream calculations, after using 1uL for the Bioanalyzer after shearing), using 0.65mL prelubricated snap cap tubes (Costar; Cat# 3206). Have ended up using a total of 3uL (includes two rounds of Bioanalyzer from today’s assays), leaving these three samples with a volume of 48uL.
Post-sonication/shearing, samples were run on High Sensitivity DNA Assay chips in the Bioanalyzer 2100 (Agilent).
All samples and volumes used are listed in the following Google Sheet (originally provided by Mackenzie Gavery):
RESULTS
Output folder:
20201027_mmag_bioanazlyer_CH05-01-21_CH07-11/
Bioanalyzer files (XAD; require 2100 Expert software to open):
- [2100 expert_High Sensitivity DNA Assay_DE72902486_2020-10-27_06-28-10.xad](https://gannet.fish.washington.edu/Atumefaciens/20201027_mmag_bioanazlyer_CH05-01-21_CH07-11/2100 expert_High Sensitivity DNA Assay_DE72902486_2020-10-27_06-28-10.xad) - [2100 expert_High Sensitivity DNA Assay_DE72902486_2020-10-27_07-41-41.xad](https://gannet.fish.washington.edu/Atumefaciens/20201027_mmag_bioanazlyer_CH05-01-21_CH07-11/2100 expert_High Sensitivity DNA Assay_DE72902486_2020-10-27_07-41-41.xad)
Images of all electropherograms (shown in a single image), as well as individual sample electropherograms, are beneath the discussion that follows.
I initially sheared the samples for an additional 10 cycles and the average fragment lengths of all three samples was still ~750bp; still a bit longer than desired. So I sheared all three samples for an additional 15 cycles (in successive 5 cycle runs).
Samples CH05-01 and CH05-21 looked great after these additional 15 cycles.
Sample CH07-11 is still a bit stubborn, but the mean fragment length is ~640bp. A tad long still, but I think it’s acceptable.
Will proceed with MDB enrichment using the MethylMiner Kit (Invitrogen) tomorrow.
Samples sheared on 20201026 + 10 additional cycles:
All samples
CH05-01
CH05-21
CH07-11
Samples sheared on 20201026 + 10 cycles + 15 additional cycles:
All samples
CH05-01
CH05-21
CH07-11
