RT-PCR primer design for ChIP

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  1. Locate the potential gene regions you believe your protein is bound to (for ChIP-seq peaks refer to Locating ChIP-seq peaks from ENCODE. Make sure this the genetic sequence is species appropriate. If not, you can use the BLAT option from the UCSC website [1] and choose the desired species from the drop-down menu.
  2. The genetic region entered for primer search should be around 400 bp.
  3. Go to NCBI primer design [2]
  4. Enter your sequence in the first box.
  5. PCR product size should be set to 70-150bp.
  6. Make sure to select the proper species in the “organism” section.
  7. Click ‘Get Primers’.
  8. Choose a primer pair that is towards the middle region, if available.
  9. Like always you should print off this page and keep it in your records, indicating the primer pair you ordered, the NM# (if one is associated) and the sequences and locations of the forward and reverse primers. Remember to record these primers in the primer database as well.
  10. Order primers from IDT [3]. It is important to indicate on the primer database and in the name on the label that these are ChIP primers so as not to be confused with normal qPCR primers.