Sam:LabNotes/Microbiome-new/2010-9-10
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MDA amplicons shearing using Misonic sonicator 3000 - with 30sec(on/off) setting[edit]
Background[edit]
- The test on 09-09-'10 showed that amplicon was over-sheared using Palsson lab protocol (pulse on 1min, pulse off 10sec)
- I noticed that this sonificator is very similar to Bioruptor I used before. After checking Bioruptor manual, I found that it needs only <5min shearing for getting 1~2Kbfragments.
- An example of Bioruptor program for 300~400bp shearing. http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Human/2009-4-25
- An example of Bioruptor setting(Diagenode) File:DNA shearing using bioruptor.pdf
Procedure[edit]
- Prepare more E.coli gDNA MDA amplicons for testing.
- Preparing UV-ed ALS, NS buffer
50uL-MDA No-SYBR recipe: 1 rxn 4.5rxn --------------------------------------------- RT-PCR grade H2O 29.0 130.5 10x RepliPhi phi-29 buffer 5.0 22.5 1mM N6 primer 2.5 11.25 RepliPhi Phi-29 (100U/ul) 2.5 11.25 dNTP (25mM) 2.0 9.0 ---------------------------------------------- 41.0 184.5/4.5=41 3uL ALS buffer + 3uL E.coli gDNA (1ng/uL) + 3uL NS buffer Transfer 41uL master mix into lysed template. Incubate at 30C for 8hr. Inactivate by 85C 3min.
- Pool the four tubes of MDA amplicons into one and quantify the yield using QUBIT-HS dsDNA
- Result: 666ng/uL in 200uL.
- Take 1ug Amplicon and dilute into 100uL H2O in a 1.5mL-tube for shearing.
Amplicon shearing test[edit]
- Directly shear the raw MDA amplicons (1ug) without purification.
- Following operation manual from 09/09/'10 with following changes on settings.
- Pulse on(30min), Pulse off(30sec), Output power(5), Temp monitoring(No). Total time(10min).
- Take out 20uL (200ng) sample for validation every 2min.
Results[edit]
- Sheared DNA were validated using 2% agarose recycled E-gel (20uL/well)(200ng/well)
File:Sam091010-E gel.jpg
- The 2min shearing with 30sec (on/off) setting can get the 1~2Kb fragment size, which is suitable for Sanger sequcncing.