CTR:Notebook/Migrant/2017/03/28: Difference between revisions

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(Autocreate 2017/03/28 Entry for CTR:Notebook/Migrant)
 
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==WIFL==
==Whole Genome Sequencing PCR-Free Library Prep for BUOW & COYE==
* Assessment of NAU WIFL extractions
* Used TruSeq Library Prep Kit


*Diluted samples 77585 (BUOW) & 1590-97213 (COYE) to 1ug of DNA in 100 uL total of Low TE
* Sonication (2017-03-28)
**BioRuptor NGS: 4 cycles of 30 seconds on, 90 seconds off, High Power
[[Image:Shear3 20150929.jpg|150px]]
1) 2uL sheared BUOW
2) 2uL 100bp low scale ladder,
3) 2uL sheared COYE
→Sheared DNA to ~400bp.
<br>
* Dried down samples to get concentrated volume of 50 uL using SpeedVac (2017-03-28)
* 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 dH<sub>2</sub>O and added 160 ul of mixture to each tube
[[Image:Sizeselect 20150930.jpg|150px]]
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:
[[Image:Finallibrary 20150930.jpg|150px]]
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 dH<sub>2</sub>O on BioAnalyzer DNA High Sensitivity Chip (2015-09-30)
**YWAR
[[Image:YWAR Bioanalyzer.png|550px]]
*WIFL
[[Image:WIFL bioanalyzer.jpg|550px]]
*[http://openwetware.org/images/5/51/SmitT-Winnie-100115-2100_expert_High_Sensitivity_DNA_Assay_DE72905009_2015-10-01_09-57-14_%282%29.pdf Bioanalyzer Results]
*Sent 10nM to Berkeley for sequencing (Illumina HiSeq 250 PER (Rapid Mode)) (2015-10-01)


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Revision as of 13:58, 28 March 2017

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Whole Genome Sequencing PCR-Free Library Prep for BUOW & COYE

  • Used TruSeq Library Prep Kit
  • Diluted samples 77585 (BUOW) & 1590-97213 (COYE) to 1ug of DNA in 100 uL total of Low TE
  • Sonication (2017-03-28)
    • BioRuptor NGS: 4 cycles of 30 seconds on, 90 seconds off, High Power

1) 2uL sheared BUOW

2) 2uL 100bp low scale ladder,

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

  • Dried down samples to get concentrated volume of 50 uL using SpeedVac (2017-03-28)
  • 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

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:

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

  • WIFL

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