Sam:LabNotes/Human/2009-4-25: Difference between revisions

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>Sam Chiang
>Sam Chiang
 
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='''Construct sequencing library - DNA shearing'''=
='''Bioruptor shearing on MDA amplicons (Bing Ren's lab)'''=


==Objectives==
==Objectives==
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*Perform shut-gun sequencing in order to clarify the concern of TIPs existence in these samples.
*Perform shut-gun sequencing in order to clarify the concern of TIPs existence in these samples.


*'''Bioruptor (Diagenode)''' for DNA shearing[http://www.diagenode.com/pages/bioruptor200.html]
*'''An example of Bioruptor setting(Diagenode) '''[[File:DNA shearing using bioruptor.pdf]]


==Procedures==
==Procedures==
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*Prepare other 4 tubes with 100uL H2O for balance in Bioruptor machine.
*Prepare other 4 tubes with 100uL H2O for balance in Bioruptor machine.
*Adjust the on and off interval (30 sec on with 30sec off)
*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.
*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.  
*Switch (clockwise)the knob to "15min" and turn on the sonification.  
*Perform three cycles of 15min sonification. Take out the samples for brief vortexing and centrifuging during the interval.
*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.
*Finished sample were stored in -20C.
==Results==
[[File:sam050410-bioruptor sheared MDA amplicon.jpg|200px]]
*Here is a Bioruptor poster which has very useful information on machine setting:
**[[File:Bioruptor DNA shearing poster.pdf]]

Latest revision as of 18:34, 12 October 2010

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