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.

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