Andrew:Notebook/C1 InTube 150525: Difference between revisions

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* I will use both standard barcoded primers as well as the Nextera transposon sequences.
* 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.
* 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.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Condition'''
| align="center" style="background:#f0f0f0;"|'''1'''
| align="center" style="background:#f0f0f0;"|'''2'''
| align="center" style="background:#f0f0f0;"|'''3'''
| align="center" style="background:#f0f0f0;"|'''4'''
| align="center" style="background:#f0f0f0;"|'''5'''
| align="center" style="background:#f0f0f0;"|'''6'''
|-
| Protocol||Transposon PCR||Transposon PCR||Transposon PCR||Transposon PCR||Standard barcodes||Standard barcodes
|-
| Sample||700 GM12878||NTC||Purified DNA||NTC||Purified DNA||NTC
|-
|
|}

Revision as of 18:29, 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 ~700 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.
Condition 1 2 3 4 5 6
Protocol Transposon PCR Transposon PCR Transposon PCR Transposon PCR Standard barcodes Standard barcodes
Sample 700 GM12878 NTC Purified DNA NTC Purified DNA NTC