Affymetrix Tiling Array Protocol

This protocol is a modified version of the RoundA/RoundB amplification used for standard ChIP on chip. The tiling arrays require more DNA (~4-5ug), so the protocol has been scaled up.

This protocol assumes a starting point of a standard ChIP experiment, with 50ul of IP DNA and 50ul input DNA, each from 50ul of lysate (if using different amounts, adjust accordingly).

Protocol:

  1. For each sample, label 3 0.2ml thin walled PCR tubes and, to each, add:
    • 7ul DNA (dilute input DNA 1:500)
    • 2ul 5X Sequenase Buffer
    • 1ul 40uM T-PCRA oligo
  2. Make reaction mix on ice:
    1X
    5X Sequenase Buffer 1 ul
    25mM dNTP 0.18 ul
    100mM DTT 0.75 ul
    500ug/ml BSA 1.5 ul
    Sequenase (13U/ul) 0.3 ul
    H2O 1.27 ul
    5 ul
  3. Place samples in PCR machine and run RoundA:
    • 2' at 94C, 2' at 8C
    • Press pause button to pause program
    • Add 5ul Reaction mix to each tube (collect micro-droplets from the sides of tubes using the pipette tip)
    • Press pause button to continue
    • Ramp to 37C over 8' (During this time prepare diluted Seuqenase)
    • 8' at 37C, 2' at 94C, 2' at 8C
    • Press pause button to pause program
    • Add 1ul diluted Sequenase (0.3ul Sequenase/0.7ul Sequenase Dilution Buffer) to each tube (again, collect micro-droplets)
    • Press pause button to continue
    • Ramp to 37C over 8'
    • 8' at 37C
    • Once the program is finished, add 35ul 1X TE to each tube to stop reaction
  4. For each sample, label 9 0.5ml thin walled PCR tubes and set up reactions:
    9X
    10X Taq PCR Buffer 90 ul
    50mM MgCl2 36 ul
    25mM dNTP 9 ul
    364mM T-PCRB oligo 30.6 ul
    Template from RoundA 135 ul
    Taq Polymerase 9 ul
    H2O 590 ul
    900 ul
  5. 100ul Reaction + 3 drops mineral oil per tube
  6. Run RoundB:
    Step: Temp: Time:
    1 92C 30"
    2 40C 30"
    3 50C 30"
    4 72C 1'
    5 Go to step one 23 times
    6 72C 10'
    7 15C hold
  7. Purify PCR reactions using Qiagen PCR kit:
    • Put all 9 tubes into a single Qiagen column sequentially (easiest if done using the vacuum manifold)
    • Elute in 50ul Elution Buffer
  8. Measure DNA concentration by using UV spec or via EtBr:
    • Mix 2ul of DNA diluted 1:10 (in H2O) with 2ul 10mg/ml EtBr (diluted 1:1000)
    • Place 2ul of this mix on a plastic petri dish
    • Make a dilution series of ssDNA (10mg/ml stock) starting from 100ng/ul down to 20ng/ul and mix with EtBr
    • Compare intensity of sample spots with dilution series on the gel imager to obtain approximate concentration
  9. Turn on PCR machine and start program WEIFRG (37C for 35'/95C for 15'/hold at 15C), when temp reaches 37C, press pause
  10. Set up fragmentation reaction in 0.2ml PCR tubes:
    • 5ug DNA
    • 5ul 10X Fragmentation Buffer
    • H2O to 50ul
  11. Add 2ul Fragmentation Mix (1.2ul 10X Fragmentation Buffer/0.2ul Fragmentation Reagent/10.6ul H2O)
  12. Place in PCR machine and press pause to continue
  13. When reaction is complete, store samples at -20C until hybridization