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 ~ | * I will also include gDNA from ~350 [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 2nd 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 2nd PCR off-chip. | ||
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| Protocol||Transposon PCR||Transposon PCR||Transposon PCR||Barcode 2nd PCR||Barcode 2nd PCR||Barcode 2nd PCR | | Protocol||Transposon PCR||Transposon PCR||Transposon PCR||Barcode 2nd PCR||Barcode 2nd PCR||Barcode 2nd PCR | ||
|- | |- | ||
| Sample|| | | Sample||350 GM12878||Purified DNA||NTC||350 GM12878||Purified DNA||NTC | ||
|- | |- | ||
| | | |
Revision as of 21:27, 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 ~350 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 2nd PCR off-chip.
Samples
- 6 samples total, 3 samples per protocol
- The products from reactions 1-3 will serve as template for reactions 4-6, similar to the C1 protocol
Condition | 1 | 2 | 3 | 4 | 5 | 6 |
Protocol | Transposon PCR | Transposon PCR | Transposon PCR | Barcode 2nd PCR | Barcode 2nd PCR | Barcode 2nd PCR |
Sample | 350 GM12878 | Purified DNA | NTC | 350 GM12878 | Purified DNA | NTC |
Protocol
- I got a fresh aliquot of 20 mg/mL Qiagen Protease from Brandon.
- Adjustment factors:
- HLS ProtQ mix --> 1.5X
- Tn5 --> 1.75X
- ProtQ --> 1.2X
- PCR --> 2.2X
- Start with 1 uL: Cells, DNA, or NTC
- Make HLS ProtQ mix for 5 reactions, 2 uL each, 1.5X: 10 uL of 7.5 mg/mL ProtQ, 45 mM Tris-HCl
- 3.75 uL 20 mg/mL ProtQ
- 1.125 uL 400 mM Tris-HCl
- 5.125 uL H2O
- Longer lysis and heat-kill: 30 min at 50 C and 30 min at 70 C.
- Make Tn5 mix for 5 reactions, 2 uL each, 1.75X: 10 uL of 0.15X Tn5 in Tn5 diluent
- 1.5 uL 1X Tn5
- 8.5 uL Tn5 diluent
- Make ProtQ mix for 5 reactions, 30 uL each, 1.2X: 150 uL of 0.24 mg/mL ProtQ, 30 mM Tris-HCl
- 1.8 uL 20 mg/mL ProtQ
- 11.25 uL 400 mM Tris-HCl
- 136.95 uL H2O
- Make PCR mix for 5 reactions, 30 uL each, 2.2X: 150 uL of 2.2X NPM, 440 nM each primer
- 99 uL 3.33X NPM
- 6.6 uL 10 uM Nextera P7 transposon
- 6.6 uL 10 uM Nextera P5 transposon
- 37.8 uL H2O