ChIP/Gene-specific PCR

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Contents

Overview

This is a protocol for 96-well format Gene Specific PCR.
Use in conjunction with chromatin immunoprecipitation to validate ChIP-chip results and determine if a protein of interest binds a specific gene in vivo.

Protocol

Designing Primers for GS-PCR

In development :)

PCR

  1. Make a stock mix plate from your two plates of GS-PCR primers (forward and reverse). For 5uM (each) stock:
    • 10 uL of 100 uM forward primer
    • 10 uL of 100 uM reverse primer
    • 180 uL of ddH2O
  2. Make dilutions of your input (wce) samples: 40 ng/uL, 20 ng/ul, and 10 ng/uL. Make dilutions of your IP samples: 10 ng/uL
    • May need to try different dilutions of wce to find a range that shows good enrichment. See comment in "Analysis". Roshan uses 45, 15, and 5 ng/uL.
  3. Make PCR master mix. Per PCR:
    • 13.6 uL of ddH2O
    • 0.2 uL of 100x dNTPs (25 mM each)
    • 2 uL of 10x ThermoPol buffer
    • 0.2 uL of Taq
    • Make 1000x and aliquot 16 uL per well into a clean PCR plate with multichannel pipettor.
  4. Add (using multichannel pipettor, according to blueprint):
    • 2 uL of primer mix (5 uM each). add blueprint
    • 2 uL of template. add blueprint
  5. Run PCR: 94°C, 2 min, 1x; 94°C, 45 sec; 58°C, 1 min; 72°C, 1 min (cycle steps 2-4, 28x); 72°C, 10 min; hold at 4°C.

Analytical Gel

  1. Pour 2.5% agarose gel in 1x TBE. Requires 500 mL for one large format gel. Set with 6 combs, spaced every-other row. (40 lanes per comb).
  2. Add 6 ul of 6x gel loading dye (kept in 4°C mini-fridge) to each 20 uL PCR reaction. Pour loading dye in trough and load with muliplex pipettor. Use yellow-box tips. Eject tips back into box for reuse when loading the gel.
  3. Load 12 uL per sample. Multiplex pipettor will load every other lane of the gel.
  4. Seal 96-well PCR plate with sticky foil cover. Store at -20°C.
  5. For ladder: 3 uL of 100 bp ladder, plus 6 uL of 6x gel loading dye.
  6. Run gel at 150 V for 2 hours. (Dye will run~2/3 the length of a row.)
  7. Prepare Sybr Gold Stain: 100 uL of Sybr Gold in 1L of 1x TBE.
  8. Cut large-format gels in half to fit in staining trays. (Earmark to differentiate).
  9. Stain gel for 1 hour, rocking at room temp.
  10. Image gel. (Be certain not to saturate the most intense peaks in the image.)

Analysis

  • Use ImageQuant for analysis of GS-PCR
  1. For each gene, check that the input (wce) dilutions show enrichment. Enrichment should be (very) roughly linear. You want to see at least two-fold enrichment in your highest dilution compared with your lowest. If not, you may need to run different dilutions of your input.
  2. Check enrichment of IP sample over input. The threshold for calling a gene enriched will be determined from the input dilutions, at least 2-fold.

Notes

References

Contacts

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