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

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>Sam Chiang
(New page: ='''DNA shearing for constructing sequencing library'''= ==Objective== *According to Illumina array analysis, the sample B3(03/31, L4N9-with Tre) and D5(04/01. N6-no Tre) were the best am...)
 
>Sam Chiang
Line 12: Line 12:
*Mearuse DNA concentration using Nanodrop.  
*Mearuse 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.
                    Sample    H2O (RT-PCR grade)
      conc.        Sample    H2O (RT-PCR grade)
   B3: 661 ng/uL    7.56      92.44
   B3: 661 ng/uL    7.56      92.44 uL
   D5: 634 ng/uL    7.88      92.12
   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 balence in Bioruptor machine.

Revision as of 05:38, 28 April 2009

DNA shearing for constructing sequencing library

Objective

  • 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.


Procedures

  • MDA amplicons were picked from samples (20uL/tube).
  • Perform 85C 3 min inactivation using PCR thermocycler
  • Mearuse 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 balence 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.