Andrew:Notebook/C1 InTube 150525: Difference between revisions
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* 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. | * 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 also include gDNA from >1000 [[http://genome-tech.ucsd.edu/LabNotes/index.php/Arichard:Samples/cells150514 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. | * 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. |
Revision as of 18:25, 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.