Protocol 37383: Probe Assay - Preb<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

>chr5:30959413-30959501 89bp GCGGGAAGCAAAAGTACAAC CTGTGAGGGAACCAAAGTGG

Mut = 89 bp

Wt = 89 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GCTCTAAGCAACAGCAACAACAACCAAAGAAACAAAAAAACAAAAAAAAACAAAAAATAAGAGCTGGAGGTGCGGGAAGCAAAAGTACAACTGAGAGAGACTAGGATTTAATgtgacttccgggctggcctgacctttgcccactttggttccctcacagcattttctgcagctagagctgatcaacgggtgcctgagcgcttccttgctgcactctcatgacacggagacacgggccaccatgaatttggcgcttgctggtgacattcttgctgccggacaggatgcccagtgtcagcttcttcgtttccaggtccatcaacagaagggcagtaaagcggagaagtcaggtgaagagctttggaaaaagtcgctctaattcttggtctccaaaaacgagagattaggtgtggggcagtggtcacattgaagtctgtacttgggtcactagaggactggtgttagttaatacagatgctcaggtccatctgagggaggtggtgcacagctgtagtctcagagcttaggagtggaggcaagaggatatttaaggctagcctggggtgcctagcaaatccctgtcttaaaaaacaacaacaaaggaatctggttcctggcaaattgtttgtgttgcaggttccaaggagcagggacctcgacagagaaagggggctcctccagcagagaagaaatcgggagcacaagttcaccccgaaggggttgaactcaaagtaaagaatttggaggcagtacagacagacttcagcaatgaaccgctgcagaaagttgtgtgcttcaaccatgataacaccctgcttgccactggaggaactgatggtcatgttcgtgtctggaaggtgtggttttaggggcttgggggggtgggtgagtgttagttgcaataacaccaaaattgtgagaatctgaatatgcattctgtgaaacttgagtggccattggggtaagagggatggggatttcagcgttctcgctgtctggtctgtaactgtcttggctgtaggtacctagcctggagaaagttctggagttcaaagcccatgaaggggagattggagatttgactttgggtcctgatggcaaggtaaggaggtggagatgtgggatgctggtgcagaaaggcagagcctattgtgtgccttgcacagtacatagatgtcctaactgtccatctttgggatctttactcctccccagctggttacagtgggctgggactttaaggcctctgtgtggcagaaggatcaactggtgacacagctacagtggcaagagaatggaccagcctcttctaatacaccataccgctaccaggcctgcaggtgtgcaggtcacagggtggctggcaggcacacctcagctgggaaatgggaaaagacagaaggagctggccatggctggagccttgttggaggttgttccaggtgtgaccagggtgcagggcacaaacatgagggctgacaggaactgaaagccttttgcctggcctccaggtttgggcaggttccagatcagctcggtgggctgcgactcttcacagtgcagataccccacaagcgcctacgtcagcccccaccctgctacctcacagcctgggacagttccaccttcttgcctcttcggaccagatcctgtggccatgaggtcatttcctgcctcagtgtcaggtgtgaagtcctactgcttgattggatggtgggcgcttgagggagctggggaatgagtcctgactcagtctctggaggaccactgttGTGGGGAGGGAGTTTGCACAAGCTTCAGAGCAATGAGCCCTCTGTTGTGTGTTACAGTGAACCCTCTGTTGTTGTATGTATGTTACAGTGAATCCTCTGTTGTATGTTACAGTGAATCCTCTGTTGTATGTATGTTACATTGAATCCTCTGTTGTGTGTTACAGTGAATCCTCTGTTGTGTTAC

This mutation is a 1660 bp deletion beginning at Chromosome 5 position 30,957,801 bp and ending after 30,959,460 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51203 GCG GGA AGC AAA AGT ACA AC Common A
51204 CTG TGA GGG AAC CAA AGT GG Wild type Reverse A
51205 ACA ACA GAG GGC TCA TTG CTC Mutant Reverse A
51206 Fluorophore-1 ACT TCC GGG CTG GCC T Quencher-1 WT Probe
51207 Fluorophore-2 ATG TGG GGA GGG AGT TTG C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51203 0.40 uM
51204 0.40 uM
51205 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.