Andrew:Notebook/C1 InTube 150525
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Troubleshooting HLS-Tn5 protocol[edit]
- AR150527_Tn5_tubeTest
Motivation[edit]
- 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[edit]
- 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[edit]
- 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
Transposon primed 1st PCR
- 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 4 reactions, 4 uL each, 1.75X: 16 uL of 0.15X Tn5 in Tn5 diluent
- 2.4 uL 1X Tn5
- 13.6 uL Tn5 diluent
- Make ProtQ mix for 4 reactions, 30 uL each, 1.2X: 120 uL of 0.24 mg/mL ProtQ, 30 mM Tris-HCl
- 1.44 uL 20 mg/mL ProtQ
- 9 uL 400 mM Tris-HCl
- 109.5 uL H2O
- Make PCR mix for 4 reactions, 30 uL each, 2.2X: 120 uL of 2.2X NPM, 440 nM each primer
- 79 uL 3.33X NPM
- 5.28 uL 10 uM Nextera P7 transposon
- 5.28 uL 10 uM Nextera P5 transposon
- 30.44 uL H2O
- Used 68 degree annealing temp
Barcoded 2nd PCR
- 3 uL 1st PCR product
- 1 uL 10 uM N7 primer
- 1 uL 10 uM S5 primer
- 5 uL 2X KAPA SYBR Fast