Andrew:Notebook/C1 InTube 150525: Difference between revisions

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* My last experiment tested HLS+ProtQ, Tn5-059, ProtQ digestion, and PCR with standard Nextera XT v2 barcoded primers.
* My last experiment tested HLS+ProtQ, Tn5-059, ProtQ digestion, and PCR with standard Nextera XT v2 barcoded primers.
* The results were negative [[http://genome-tech.ucsd.edu/LabNotes/index.php/Andrew:Notebook/C1_OpenApp_150429 Troubleshooting the Tn5 titration experiment]]
* The results were negative: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Andrew:Notebook/C1_OpenApp_150429 Troubleshooting the Tn5 titration experiment]]
* This indicates that either the protease or Tn5 died, so I am going to make fresh aliquots and repeat.
* I will also include gDNA from >1000 GM12878 cells extracted with HLS + G-HCl and purified with Ampure XP.
* I will use both standard barcoded primers as well as the Nextera transposon sequences.
* If the transposon sequences work, I can do the Tn5 titration on chip with a back-loaded Tn5 curve and common PCR mix, followed by barcoded PCR off-chip.

Revision as of 18:09, 26 May 2015

Troubleshooting HLS-Tn5 protocol

  • C1_InTube_150525

Motivation

  • My last experiment tested HLS+ProtQ, Tn5-059, ProtQ digestion, and PCR with standard Nextera XT v2 barcoded primers.
  • The results were negative: [Troubleshooting the Tn5 titration experiment]
  • This indicates that either the protease or Tn5 died, so I am going to make fresh aliquots and repeat.
  • I will also include gDNA from >1000 GM12878 cells extracted with HLS + G-HCl and purified with Ampure XP.
  • I will use both standard barcoded primers as well as the Nextera transposon sequences.
  • If the transposon sequences work, I can do the Tn5 titration on chip with a back-loaded Tn5 curve and common PCR mix, followed by barcoded PCR off-chip.