CTR:Notebook/Migrant/2015/09/29

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Whole Genome Sequencing PCR-Free Library Prep for YWAR & WIFL

  • Used TruSeq Library Prep Kit
  • Diluted samples 77585 (YWAR) & 1590-97213 (WIFL) to 1ug of DNA in 100 uL total of Buffer AE
  • Sonication (2015-09-29)
    • BioRuptor NGS: 4 cycles of 30 seconds on, 90 seconds off, High Power

Shear3 20150929.jpg

1) 2uL sheared YWAR

2) 2uL 100bp low scale ladder,

3) 2uL sheared WIFL →Sheared DNA to ~400bp.

  • Dried down samples to get concentrated volume of 50 uL using SpeedVac (2015-09-29)
  • Clean-up fragmented DNA (2015-09-30)
  • End Repair and Size Selection- edited to size select for 400bp (2015-09-30)
    • Made a diluted bead mixture with the ratio of 105ul beads: 79ul dH2O and added 160 ul of mixture to each tube

Sizeselect 20150930.jpg

1) 2uL YWAR

2) 2uL 100bp low scale ladder,

3) 2uL WIFL

  • Adenylate 3' Ends (2015-09-30)
    • Ran on JOHN thermocycler under program: SDCRKAD, Block B
  • Ligate Adapters (2015-09-30)
    • Assigned Adapter 2 to YWAR and Adapter 4 to WIFL
  • Gel of Final Library:

Finallibrary 20150930.jpg

1) 2uL YWAR

2) 2uL 100bp low scale ladder,

3) 2uL WIFL

  • Picogreen:
    • YWAR: 7.42 ng/μL
    • WIFL: 6.72 ng/μL
  • Run 1μL of sample + 2μL of dH2O on BioAnalyzer DNA High Sensitivity Chip (2015-09-30)
    • YWAR

YWAR Bioanalyzer.png

  • WIFL

WIFL bioanalyzer.jpg

  • Sent 10nM to Berkeley for sequencing (Illumina HiSeq 250 PER (Rapid Mode)) (2015-10-01)