Testing Sonication for MBD

Sonication Tests with Q800R3 Sonicator

Using two samples to test optimal time and intensity for shearing coral DNA for MBD library prep

Samples:
Acropora sperm # 221 (High molecular weight DNA)
Montipora soft homogenization # 1431 (smeared DNA)

Using 1µg of DNA in 50µl of ultra pure water in these tubes

Sample Vol DNA to 1µg Vol H20 to 50µl Tube #
221 7.69µl 42.31µl 9
1431 5.21µl 44.79µl 10
221 7.69µl 42.31µl 11
1431 5.21µl 44.79µl 12
221 7.69µl 42.31µl 13
1431 5.21µl 44.79µl 14
  1. Used QSonica protocol to set up the sonicator
  2. Set the amplitude to 25%
  3. Set the pulse to be 15 seconds on and 15 seconds off
  4. Used 3 different time setting to test what would give us shearing to about 500 basepairs (1 minute setting, 2.5 minute setting, and 1.5 minute settings)
  5. A cycle is defined as 15 seconds on and 15 seconds off (so 30 seconds total)
  6. 1 minute and 2.5 minute settings were done first, visualized, and then we decided to try in the middle with a 1.5 minute setting
Sample Tube # Time Setting # of Cycles
221 9 1 min 4 cycles
1431 10 1 min 4 cycles
221 11 2.5 min 10 cycles
1431 12 2.5 min 10 cycles
221 13 1.5 min 6 cycles
1431 14 1.5 min 6 cycles
  1. Visualization with D5000 tapes for the TapeStation 4200 (protocol)

1 minute setting and 2.5 minute setting results first

1.5 minute setting results second

1.5 minute setting seems to be the best option for getting around 500 bp, for both starting HMW and smeared, even though there are differences

Written on July 30, 2019