ChIP/Gene-specific PCR

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.

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:
 * 2) * 10 uL of 100 uM forward primer
 * 3) * 10 uL of 100 uM reverse primer
 * 4) * 180 uL of ddH2O
 * 5) 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
 * 6) * 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.
 * 7) Make PCR master mix. Per PCR:
 * 8) * 13.6 uL of ddH2O
 * 9) * 0.2 uL of 100x dNTPs (25 mM each)
 * 10) * 2 uL of 10x ThermoPol buffer
 * 11) * 0.2 uL of Taq
 * 12) * Make 1000x and aliquot 16 uL per well into a clean PCR plate with multichannel pipettor.
 * 13) Add (using multichannel pipettor, according to blueprint):
 * 14) * 2 uL of primer mix (5 uM each). add blueprint
 * 15) * 2 uL of template. add blueprint
 * 16) 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.

Contacts

 * cmc