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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.
Designing Primers for GS-PCR
In development :)
- 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
- 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.
- 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.
- Add (using multichannel pipettor, according to blueprint):
- 2 uL of primer mix (5 uM each). add blueprint
- 2 uL of template. add blueprint
- 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.
- 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).
- 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.
- Load 12 uL per sample. Multiplex pipettor will load every other lane of the gel.
- Seal 96-well PCR plate with sticky foil cover. Store at -20°C.
- For ladder: 3 uL of 100 bp ladder, plus 6 uL of 6x gel loading dye.
- Run gel at 150 V for 2 hours. (Dye will run~2/3 the length of a row.)
- Prepare Sybr Gold Stain: 100 uL of Sybr Gold in 1L of 1x TBE.
- Cut large-format gels in half to fit in staining trays. (Earmark to differentiate).
- Stain gel for 1 hour, rocking at room temp.
- Image gel. (Be certain not to saturate the most intense peaks in the image.)
- Use ImageQuant for analysis of GS-PCR
- 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.
- 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.