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1. Label the appropriate number of PCR tubes or PCR plates compatible with the model of real-time PCR machine to be used. PCR reactions should be performed in duplicate and should include a tube with no DNA to control for contamination, and a serial dilution of a 2% total input chromatin DNA (undiluted, 1:5, 1:25, 1:125), which is used to create a standard curve and determine amplification efficiency.2. Add 2 μl of the appropriate ChIP DNA sample to each tube or well of the PCR plate.
3. Prepare a master PCR reaction mix as described below. Add enough reagents for two extra reactions to account for loss of volume. Add 18 μl of the master PCR reaction mix to each PCR reaction tube or well of the PCR plate.
Reagent Volume for 1 PCR Reaction (20 μl)
Nuclease-free H2O 6 μl
5 μM SimpleChIP® Primers 2 μl
2X SYBR® Green Reaction Mix 10 μl
4. Start the following PCR reaction program:
a. Initial Denaturation: 95°C for 3 min.
b. Denaturation: 95°C for 15 sec.c. Anneal and Extension: Primer-specific temp. for 60 sec.
d. Repeat steps b and c for a total of 40 cycles.
5. Analyze quantitative PCR results using software provided with the real-time PCR machine.