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. | ||
===Samples=== | |||
* 6 samples total, make for 8 reactions. | |||
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===Protocol=== |
Revision as of 18:30, 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.
Samples
- 6 samples total, make for 8 reactions.
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 |