Difference between revisions of "RT-PCR primer design for ChIP"
From Bridges Lab Protocols
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| − | # Locate the potential gene regions you believe your protein is bound to (for | + | # 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 [https://genome.ucsc.edu/index.html] and choose the desired species from the drop-down menu. |
# The genetic region entered for primer search should be around 400 bp. | # The genetic region entered for primer search should be around 400 bp. | ||
# Go to NCBI primer design [http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?ORGANISM=9606&INPUT_SEQUENCE=NM_001618.3] | # Go to NCBI primer design [http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?ORGANISM=9606&INPUT_SEQUENCE=NM_001618.3] | ||
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# 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. | # 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 [https://www.idtdna.com/site]. 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. | # Order primers from IDT [https://www.idtdna.com/site]. 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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| + | [[ Category: Immunoprecipitation ]] | ||
| + | [[ Category: Transcription ]] | ||
| + | [[ Category: PCR ]] | ||
Latest revision as of 14:24, 21 August 2016
- 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.
- The genetic region entered for primer search should be around 400 bp.
- Go to NCBI primer design [2]
- 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]. 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.
