Protocol 32604: Probe Assay - Pmpca<em1(IMPC)J>
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

>chr2:26390853+26390950 98bp CCTAGGAAGCCCTGAGTGAG ACTGCCCACAGTGAAGCTCT

Mut= 95 bp

Wt= 98 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 

ATGCTAAGCTAAACAGGCAGGCTGCTGCTGGCTATCTGAAGCTGGGCATGTGTCCATGTGAAACCAGTCTTCATTTAGACCAGCTGGTTATTTGTCCAGGCCTAGGAAGCCCTGAGTGAGAGACttcctctgtgaagagaggagaaccagtttcctctggttccaaagtatctcatggagagcttcactgtgggcagttgtggctcccagaaggccttcacacccaagaaagaggtagtggtagtgtctttgctggcatgaagggcctcagtgactgagttgttatctggtgtaaaccatgcacctgctattgtggcttatttcctttcagagacaccaccatgtatgctgtgtctgctgacagcaaaggcttggacactgtggtggatctgttggctgatgtggttctgcacccccgactgacaggtgtggctctgaggctgggtggtcaggaggagctttggaagggctataacttcctgtggggaggcactgttagagccgggcactcatgtgccaatcattggagagtgccagtgctgaaacaagcctgcagtgttctacacaatgtttcatcctttaatatatctaaacgagaccttaaagggtttactaaagttctgctcagaaatgtccctcatcccgtgtgTAAGCATGAATGCCATGCCTACAGAAGTTTCTCTTGCCTGGTCTCCTTTATCCTCCGAGTCTTTGAGTCCTGTTAGGATGGGAGAGGGCGCAGCA

This mutation is a 525 bp deletion beginning at Chromosome 2 position 26,390,877 bp and ending after 26,391,401 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39535 CCT AGG AAG CCC TGA GTG AG Common A
39536 ACT GCC CAC AGT GAA GCT CT Wild type Reverse A
39537 AGG ACT CAA AGA CTC GGA GGA Mutant Reverse A
39538 Fluorophore-1 TCT GTG AAG AGA GGA GAA CCA GTT Quencher-1 WT Probe
39539 Fluorophore-2 AGA GAC TAA GCA TGA ATG CCA TG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39535 0.40 uM
39536 0.40 uM
39537 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.