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Testing Sonication for MBD |
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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 |
- Used QSonica protocol to set up the sonicator
- Set the amplitude to 25%
- Set the pulse to be 15 seconds on and 15 seconds off
- 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)
- A cycle is defined as 15 seconds on and 15 seconds off (so 30 seconds total)
- 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 |
- Visualization with D5000 tapes for the TapeStation 4200 (protocol)
1 minute setting and 2.5 minute setting results ![first]({{ site.baseurl}}/images/first-sonication-test.png "first")
1.5 minute setting results ![second]({{ site.baseurl}}/images/second-sonication-test.png "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