Protocol 31547: Probe Assay - Map7d1<em1(IMPC)J> Probe
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

Mutant=  144 bp

Wild Type = 150 bp

>chr4:126240200-126240349 150bp CTCATACACCCAGGAAGTGAC CCCCAATCCTAAGCAATTC

Sequence

Wt Sequence (deleted region in lower case):

ATGAAAGCACCAAGATCAGGACCCTCAGTACTAGGGAGACCCTGCCCAGGAATCCCAGGCAAAGAAGAGTCCCACCCATCCTCAGAGAAGCAACTCATACACCCAGGAAGTGACTCAAGTCCatggggaaaaaaaaagctctccaagaactcggatagggatctatcacacataggcttgactcacagccaagtcaggaactttggttgcctgtagtgagtcaggaattgcttaggattggggaggactttctccaggtatggtggctgatcctgagcccagatctttttcccattggcccccaggagcgctatgaagcagcaatccagcggtcagtgaagaagacgtgggctgagatccggcaacagcgctggtcctgggcaggggccctgcaccacagctcccctggacgtaagaccagtgagtgggctggaagtgctggcagacGGGCGGGTGGGTAGGTGGGCAGGGCTGTGGACAGGAACACTATACACAAggcGGTCGGTCGTTCACTACTCCCTGCACCAGGTGTGTCTTGCTTCGTGTGACTTGTGTCTTCTGGAAT

 

 

325 bp deletion beginning at Chromosome 4 negative strand position 126,240,320 bp and ending after 126,239,996 bp (GRCm38/mm10). This mutation deletes ENSMUSE00000364976 (exon 5) and 210 bp of flanking intronic sequence including the splice acceptor and donor. In addition there is a 3 bp deletion (GGC) 59 bp after the 325 bp deletion that will not alter the results of the exon deletion

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37127 CTC ATA CAC CCA GGA AGT GAC Common A
37128 CCA GAA GAC ACA AGT CAC ACG Mutant Reverse A
37129 CCC CAA TCC TAA GCA ATT C Wild type Reverse A
37130 Fluorophore-1 CAG GGC TGT GGA CAG GAA Quencher-1 MUT Probe
37131 Fluorophore-2 TCG GAT AGG GAT CTA TCA CAC ATA Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37127 0.40 uM
37128 0.40 uM
37129 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.