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

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>Sam Chiang
>Sam Chiang
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='''DNA shearing for constructing sequencing library'''=
='''DNA shearing for constructing sequencing library'''=


==Objective==
==Objectives==
*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.
*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 clearify the concern of TIPs existance 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]
*'''Bioruptor (Diagenode)''' for DNA shearing[http://www.diagenode.com/pages/bioruptor200.html]
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*MDA amplicons were picked from samples (20uL/tube).
*MDA amplicons were picked from samples (20uL/tube).
*Perform 85C 3 min inactivation using PCR thermocycler
*Perform 85C 3 min inactivation using PCR thermocycler
*Mearuse DNA concentration using Nanodrop.  
*Measure DNA concentration using Nanodrop.  
**Transfer 5ug sample into 1.5-mL microcentrifuge tubes and add water into 100 uL.
**Transfer 5ug sample into 1.5-mL microcentrifuge tubes and add water into 100 uL.
       conc.        Sample    H2O (RT-PCR grade)
       conc.        Sample    H2O (RT-PCR grade)
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   D5: 634 ng/uL    7.88      92.12 uL
   D5: 634 ng/uL    7.88      92.12 uL


*Prepare other 4 tubes with 100uL H2O for balence 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)
*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.

Revision as of 05:45, 28 April 2009

DNA shearing for constructing sequencing library

Objectives

  • 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.
  • Bioruptor (Diagenode) for DNA shearing[1]

Procedures

  • 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)
  • 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. Take out the samples for brief vortexing and centrifuging during the interval.
  • Finished sample were stored in -20C.