Protocol 37848: Probe Assay - Sec31b<em1(IMPC)J> Alternate 3
Version 4.0

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

 


>chr19:44536065-44536156 92bp AATGTGACCCACGTCCTTTC GGATTAAAGTCCAAGGCTCTG

 

Mut = 132 bp

Wt = 157 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 AGTCAGTGCTCTTAATCGATGAGCCATCTCTCCAGCCTGGAGTATTTTTTTTTTCTTTCCTAAAGAAAGGGCAAGTCTGGGCAGGGGAGACTTCCCTTCTGGGTCTGGAGCAAGAAGAAAATAGACAAGCAGATACAGGCTTGGGCTGGGGAAGCCTGCATGATAGGAGAagctggcgaagatgtctgtcctctgcataatgacatgtgcctgcctcggtgctgagactgattctttgcctttgtaggtttcacaagctaatatggggaagctttggcagtgggcttctggaaaactccggggttattgctggtggaggagacaacggcacactgactctatacaatgtgacccacgtcctttcttcagggaaggagcccctgattgcccagaaacagaagcacactggagctgtcagagccttggactttaatcctttccaggtactgtgtcttagctgaccagacctggtcaaggcatgacgacgcatgtctgaatggaagcattctcttgtggccttcaggctgtgcctctgaactgtaatgggagaggccctgggtctaccaaatgcaagcctcctctttctccttttctgaggtgggagtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtttgggggtaggtctttagaaatatcttttattcacacagggcaatctcctggcctcaggagccagtgactctgagatcttcatttgggatttgaaccacctgagtgtcccaatgacacctggacccaagtcacaggtgaggggctccctcctccctctgacctggggctgcctcttagggtgtacagccaggttttcccaggtaaagtttggaattgtggagccagccgagtaacagagatttccagctttaccactggagggcaataaaaccccacagatggacaggtcacctagtagtcaggagAGAGGGCAGAGCAGGTGGTTCACGGTGGTCAGTGCTGTTCAAAGAATTCCTGGAAAGAAGTGAAGGGCAAGGGGGGACAGAAGACACCAAGACAGGCAAGGAGTGATATGTATCATAGT

This mutation is a 697 bp deletion beginning at Chromosome 19 position 44,535,584 bp and ending after 44,536,280 bp (GRCm38/mm10).

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49662 AAT GTG ACC CAC GTC CTT TC Wild type Forward A
49666 Fluorophore-1 AGG GAA GGA GCC CCT GAT TG Quencher-1 WT Probe
51671 GGA GCA AGA AGA AAA TAG ACA AGC A Mutant Forward A
51672 Fluorophore-2 ACA GGC TTG GGC TGG GGA A Quencher-2 MUT Probe
52321 TCC ATT CAG ACA TGC GTC GT Wild type Reverse A
52322 ACT GAC CAC CGT GAA CCA C Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49662 0.40 uM
51671 0.40 uM
52321 0.40 uM
52322 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.