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

  1. Start with 1 uL: Cells, DNA, or NTC
  2. Make HLS ProtQ mix for 5 reactions, 2 uL each, 1.5X: 10 uL of 7.5 mg/mL ProtQ, 45 mM Tris-HCl
    1. 3.75 uL 20 mg/mL ProtQ
    2. 1.125 uL 400 mM Tris-HCl
    3. 5.125 uL H2O
    4. Longer lysis and heat-kill: 30 min at 50 C and 30 min at 70 C.
  3. Make Tn5 mix for 4 reactions, 4 uL each, 1.75X: 16 uL of 0.15X Tn5 in Tn5 diluent
    1. 2.4 uL 1X Tn5
    2. 13.6 uL Tn5 diluent
  4. Make ProtQ mix for 4 reactions, 30 uL each, 1.2X: 120 uL of 0.24 mg/mL ProtQ, 30 mM Tris-HCl
    1. 1.44 uL 20 mg/mL ProtQ
    2. 9 uL 400 mM Tris-HCl
    3. 109.5 uL H2O
  5. Make PCR mix for 4 reactions, 30 uL each, 2.2X: 120 uL of 2.2X NPM, 440 nM each primer
    1. 79 uL 3.33X NPM
    2. 5.28 uL 10 uM Nextera P7 transposon
    3. 5.28 uL 10 uM Nextera P5 transposon
    4. 30.44 uL H2O
  • Used 68 degree annealing temp

Barcoded 2nd PCR

  1. 3 uL 1st PCR product
  2. 1 uL 10 uM N7 primer
  3. 1 uL 10 uM S5 primer
  4. 5 uL 2X KAPA SYBR Fast

Gels[edit]

File:AR150527 gels Slide1.jpg

File:AR150527 gels Slide2.jpg