Protocol 37846: Probe Assay - Sptb<em1(IMPC)J> Alternate 1
Version 2.0

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

>chr12:76620341-76620465 125bp AAATACTTCTCAGCAACAGGAGTC GCACAGAGAGTTAGAGGTGACAGA

Mut = 114 bp

Wt = 125 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

GGCCCTTGTCATAAATAAGAGTGTTCATTACTTCCTGGGAATCTGTAACTGAGCTTTTTAAAATCTCATGTCATGTTATGCATTTCTACAAGAACTGACTCAACTTTCAATCTCCCtcatttctgaagtgtggtgggagccagttataaagaatccgtgctgtcctttgagtaactgagaatccatggctccagtcccacagctccttgaagggttagagacatagctcagtggtcgagcatttgcctaccacacacaaggtcgccaagaccatgaaaaatggaagtttgacattgtgtggtcctgatctgaaatttgcctataaataaatcccttctttgccttcctcctcctattcctccagggtatcaaccttgtgacccccaggtcatccaggaccgtgtcagccacctagagcagtgcttcagtgaactgagcaacatggcagctggacggaaggctcagctggagcagtctaagcgactctggaagttcttctgggagatggatgaggccgaaagctggatcaaggagaaggagcagatctactcctccctggactacggcaaggacctgaccagcgtgctcatcttacagcgcaagcacaaggcctttgaggatgagctccgtgggctggacgcccatctgaaacagatcttccaggaggctgacgacatggttgcacaaaagcagtttggacacccacagatcgagacccgtgtcaaagaagtgtcagctcagtgggaccacctgaaggagctggctgcctttcgtaagaaagacctgcaggatgcggaaaacttcttccagttccagggtgatgcagacgacctgaaggcttggctgcaagacgctcaccggctgctctctggtgaagatgtagggcaagatgaaggagccacgagggccctgggaaagaagcacaaggagttcctggaggagctggaggagagccgaggggtgatggagcacttggagcaccaagcccagggcttccccgaggagtttcgagattccccagatgtaacaaaccgactccaggcactcaggaagctctaccagcaggtactgacccaggcagaattgcgtggacacaagctgcaggaagccctggacttgtacaccgtgtttggggagtcagatgcctgtgagctgtggatgacggagaaggggaagtggctggaccagatggatatcccaaacacgctggaggatctggaggtggtgcagcacaggtcagacagctcagccttgctcatccccacccgagagctgagggtcacctcccactgctagaccctcctctgttacatagtgcaggcatcaggaattgagttctggtctgactccggggcctccgattcttcacccactggcacttcatagttctggagacaaaggggttttttaaaaaatatattattaaaatacttctcagcaacaggagtctttactcagatgaattctgagcattgggatacaggaAGAGAGGAGGTGCTGTGGACATGGGGCTGAGAGGCTGGGATTCTGTCACCTCTAACTCTCTGT

This mutation is a 1369 bp deletion beginning at Chromosome 12 position 76,620,406 bp and ending after 76,621,774 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
50313 GCA CAG AGA GTT AGA GGT GAC AGA Common A
50314 AAA TAC TTC TCA GCA ACA GGA GTC Wild type Forward A
50316 Fluorophore-1 CTC AGA TGA ATT CTG AGC ATT GGG Quencher-1 WT Probe
50317 Fluorophore-2 CAA CTT TCA ATC TCC CAC AGA GAG G Quencher-2 MUT Probe
52319 GTC ATG TTA TGC ATT TCT ACA AGA AC Mutant Forward A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
50313 0.40 uM
50314 0.40 uM
52319 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.