RT-PCR primer design for ChIP
- 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] ( attr(href) ) and choose the desired species from the drop-down menu.
- The genetic region entered for primer search should be around 400 bp.
- Go to NCBI primer design [2] ( attr(href) )
- Enter your sequence in the first box.
- PCR product size should be set to 70-150bp.
- Make sure to select the proper species in the “organism” section.
- Click ‘Get Primers’.
- Choose a primer pair that is towards the middle region, if available.
- 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.
- Order primers from IDT [3] ( attr(href) ). 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.
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