Answers.Body: <p>Primer-blast is okay, but I presume these are lab mice so the variation is low, thus  you can just use Primer3. SNPs are detected via TaqMan <a href="https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/snp-genotyping-taqman-assays.html?cid=gsd_pcr_sbu_r03_co_cp1491_pjt9621_gsd00000_0se_gaw_rs_pur_&amp;ef_id=Cj0KCQiA7OqrBhD9ARIsAK3UXh1z5e06QTnP0pbSLGufHAvSHGh9vAvaNNLLNRoDiQVRnR9zgH6thosaAn1mEALw_wcB:G:s&amp;s_kwcid=AL!3652!3!606132911456!e!!g!!taqman%20snp%20genotyping%20assay!17574808757!139287689898&amp;gad_source=1&amp;gclid=Cj0KCQiA7OqrBhD9ARIsAK3UXh1z5e06QTnP0pbSLGufHAvSHGh9vAvaNNLLNRoDiQVRnR9zgH6thosaAn1mEALw_wcB" rel="nofollow noreferrer">here</a>, i.e. a separate probe that sits over the mutation site, it will detect heterozygosity via qPCR signal (half strength than homozygous).</p>
  
Answers.ContentLicense: CC BY-SA 4.0
Answers.CreationDate: 2023-12-14T22:25:19.080Z
Answers.LastActivityDate: 2023-12-14T22:25:19.080Z
Answers.UserDisplayName: M__
Body: <p>My PI has asked me for advice on making a PCR screen to identify mice from a particular Cre line.  We have multiple Cre lines in the colony so it can't just be for mice with a copy of Cre in their genome the primer. The insertion site is known for the mutation,  is there a way to use blast or some other search tool to pull the sequences we should base our primers on?</p>
  <p>We also want to identify which mice are hetero vs homozygous.  Are there particular kinds of PCR we should use or will a well designed primer enable this?</p>
  <hr />
  <p><strong>Details</strong></p>
  <p>The strain:  <a href="https://www.jax.org/strain/004146" rel="nofollow noreferrer">https://www.jax.org/strain/004146</a></p>
  <p>I was thinking of using this tool:  <a href="https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome" rel="nofollow noreferrer">https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome</a></p>
  <p>The strain reference from Jax, not sure the recommended <strong>cre</strong> primers are going to be sufficient for what we want.  So I'm trying to find the FASTA file for this strain in the region of interest, then I can throw that into the BLAST tool for primer design I listed above.  Not sure where to search for the FASTA for this strain though. Strain information: <a href="https://www.informatics.jax.org/reference/J:66884" rel="nofollow noreferrer">https://www.informatics.jax.org/reference/J:66884</a></p>
  <p>Jax recommends this protocol so I suggested it:  <a href="https://www.jax.org/Protocol?stockNumber=004146&amp;protocolID=20627" rel="nofollow noreferrer">https://www.jax.org/Protocol?stockNumber=004146&amp;protocolID=20627</a></p>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2023-12-14T21:21:37.797Z
Comments.Text: Jax recommends this protocol so I suggested it:  https://www.jax.org/Protocol?stockNumber=004146&protocolID=20627
Comments.UserDisplayName: Angus Campbell
Tags: <primer><pcr><qpcr>
Title: PCR primers for detecting hetero vs homozygous and WT mice in our colony
ThreadText: # PCR primers for detecting hetero vs homozygous and WT mice in our colony
  
  (Asked by: username_0 on 2023-12-14)
  
  My PI has asked me for advice on making a PCR screen to identify mice from a particular Cre line. We have multiple Cre lines in the colony so it can't just be for mice with a copy of Cre in their genome the primer. The insertion site is known for the mutation, is there a way to use blast or some other search tool to pull the sequences we should base our primers on?
  
  We also want to identify which mice are hetero vs homozygous. Are there particular kinds of PCR we should use or will a well designed primer enable this?
  
  ---
  
  **Details**
  
  The strain: <https://www.jax.org/strain/004146>
  
  I was thinking of using this tool: <https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome>
  
  The strain reference from Jax, not sure the recommended **cre** primers are going to be sufficient for what we want. So I'm trying to find the FASTA file for this strain in the region of interest, then I can throw that into the BLAST tool for primer design I listed above. Not sure where to search for the FASTA for this strain though. Strain information: <https://www.informatics.jax.org/reference/J:66884>
  
  Jax recommends this protocol so I suggested it: <https://www.jax.org/Protocol?stockNumber=004146&protocolID=20627>
  
  **Tags:** primer, pcr, qpcr
  
  > **Comments:**
  > **username_0**: Jax recommends this protocol so I suggested it:  https://www.jax.org/Protocol?stockNumber=004146&protocolID=20627  
  
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  ## 1 answers
  
  ### Answer by username_1 on 2023-12-14. Score: 1 | Accepted answer
  
  Primer-blast is okay, but I presume these are lab mice so the variation is low, thus you can just use Primer3. SNPs are detected via TaqMan [here](https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays/snp-genotyping-taqman-assays.html?cid=gsd_pcr_sbu_r03_co_cp1491_pjt9621_gsd00000_0se_gaw_rs_pur_&ef_id=Cj0KCQiA7OqrBhD9ARIsAK3UXh1z5e06QTnP0pbSLGufHAvSHGh9vAvaNNLLNRoDiQVRnR9zgH6thosaAn1mEALw_wcB:G:s&s_kwcid=AL!3652!3!606132911456!e!!g!!taqman%20snp%20genotyping%20assay!17574808757!139287689898&gad_source=1&gclid=Cj0KCQiA7OqrBhD9ARIsAK3UXh1z5e06QTnP0pbSLGufHAvSHGh9vAvaNNLLNRoDiQVRnR9zgH6thosaAn1mEALw_wcB), i.e. a separate probe that sits over the mutation site, it will detect heterozygosity via qPCR signal (half strength than homozygous).
  
  
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