Sam:LabNotes/Human/2009-4-25
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Bioruptor shearing on MDA amplicons (Bing Ren's lab)[edit]
Objectives[edit]
- According to Illumina array analysis, the sample B3(03/31, L4N9-with Tre) and D5(04/01. N6-no Tre) were the best amplicons in single cell amplification.
- Perform shut-gun sequencing in order to clarify the concern of TIPs existence in these samples.
- An example of Bioruptor setting(Diagenode) File:DNA shearing using bioruptor.pdf
Procedures[edit]
- MDA amplicons were picked from samples (20uL/tube).
- Perform 85C 3 min inactivation using PCR thermocycler
- Measure DNA concentration using Nanodrop.
- Transfer 5ug sample into 1.5-mL microcentrifuge tubes and add water into 100 uL.
conc. Sample H2O (RT-PCR grade) B3: 661 ng/uL 7.56 92.44 uL D5: 634 ng/uL 7.88 92.12 uL
- Prepare other 4 tubes with 100uL H2O for balance in Bioruptor machine.
- Adjust the on and off interval (30 sec on with 30sec off)
- This program is designed to obtain 300~400bp fragments
- Place the sample tubes in tube holder and check the cooling water is circulating normally.
- Switch (clockwise)the knob to "15min" and turn on the sonification.
- Perform three cycles of 15min sonification (45min in total). Take out the samples for brief vortexing and centrifuging during the interval.
- Finished sample were stored in -20C.
Results[edit]
File:Sam050410-bioruptor sheared MDA amplicon.jpg
- Here is a Bioruptor poster which has very useful information on machine setting: