Protocol 35486: Probe Assay - Nkpd1<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

>chr7:19525099+19525241 143bp GCTATCTGACCCACCTACTTCC GCGTCTGCCCTTCATCATC

Mut= 141 bp

Wt= 143 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 CATTCATCTGTTTCTCTATCCATTAATATTCTTGTACCGGGTCATTTCATTCATTCCTGGAGTGGCCACAACTTAAGGCCCTGTCTTTCTTGTGACATCCTGGCTGCCTCACCCTCTTGCCTGGTCCTGGCTCTTTCTACCTCCCTCCCAtcctttctcagggaggtccctacttttgtcctagacctccaggcctcttccttcccacctacatgcaccaccagcctctccccgacccgtctccactgacgccttgcctcctctcccccagcgctgatgcagcaggaggccgcgcagcgtgagagcgaggagctgcagcgcgtgcagtggcagccacgtcgcgtgcgcggctggggcgtcccgaagctgctgtggttcctggtgtttctgcagcccgtgatcacggagctgcacctgcggcgcaggaatgtgcgcttcctgttcatccgcttcagcgcctggcagtacgcgggcaccgacaagctgtgggccgggctggtgaccacgctgtgtgagggcatccgccaccactacggcgcgctgccctttagcgtgtactcggtgctaggcaacaagccctgtggcccgcgtgatggcctctgccaacgcgagtggcactgccgacgtcgcgtgtgcctggcgctgctggcgctgctggccgcgctgtgcctgggtgtggggctgctgtacctgtcgctgggaggccacgcgccgggccacggagagcgtggtgtgctcaaagcgctgggcggcgcggccaccacgctatccggctcagggctgctcatggccgtgtactccgtgggcaagcatctgttcgtgagccagcgcaagaagatcgagcgcctggtgtcgcgtgagaagtttggcagccagttgggcttcatgtgcgaggtgaagaaggaggtggaactgctcacggatttcctgtgcttcctggagatctaccagcgacgccggctgcgtgtggtcctcgaggtcactgggttggacacgtgctacccagagcgcgtggtgggcgtcctaaacgccatcaacactctgctgtccgacagccacgctcccttcatcttcatcctggtggtggaccccagcattctggccgcgtgcctggagagcgcgggtaacatgaagggcacagcagacaacggctacctgttcctcaaccgcacagtcacgctacccttctccgtgccgattatgggccgtcgcaccaagttgcagttcctgcacgatgcggtgcgtagccgggacgatctgctgttccgggaattgacgatcaagctacagccgcagagccccgggaacttgggcgcaggtgagggtactcagctgctggcggtggagacgcagggggacgccgagcgcacacagggccgcgtggacgcggaggcggcgcggcgtatccaggaggcgctgtgctgcctgcatgacgagggtgattgcctgtatgagtacgtgcccgacaacgtggtgtccatgcggcgcattgtcaacacggtgcccatcaccgtgcgcctgttgcagcagcagcagcagcaacagcccgaccgcgtgggacccacaccgcgccacgcggtggcctgggtcgtcctcgctaaccagtggccttgtcgcctcagctgggtgctgcaatgcctggaggaccggcagcaggcagggggcgcgccagaggggcgctcgcgtctctgggatgtcttctgcgacaatagtcgcgagctgcacaccatgaccaaggccttgcagaacgtactggacctggatggcgaccctgagctttttgagcgctttctaggcactgatttccccttcaccgtggccgaggcgcagagtttgctgcgctgcacagtcaacctggaccattccatccgccgccgcatgggcctcatcagggccgtcagcgcgctcaagccgcccagcccacccaagtccccatcccaggacggtccccaggccagtcccagagccattatcgccgcaggcacgtcccatgcgggccaaggatcaggacacagtaaagaggcacatcagacccgggaccggacccatgggggcaagccaaggccgatggcctaagtccttaaatttcaggagcgtagaggctgtttgggtgtgggatggagaacaccagccacgggagacagcaacatccctcctgacctgatgagggtgtgagggcaagaggggctcaaaaccaatggggtatggccacacaccctgatgactttggaagccaaggggaagcaggtactactagcttcctggagtaggcagcttctacaccagtgtctgagtgagcagaggagtcagtgaggaacacagagtcaaagaagcaataaaggaagctgtggaaagccagggctgtctttcatgtcctggatttctggctaggtgagaatgctatctgacccacctacttcctcctcagggagccaggcctctggactgcaccctggcagctgaagttggcatcccAGGTGGTAGGAGGCTTGAGGGGGTGGTGGGTGAGGCTGCGGGGGCAGGGATGATGAAGGGCAGACGCCAAGTTGACTCAGGCAAAGGGGAGCACCCAGGCCTCAGGGGATGCCAGGCCTTTGTCCCCACACCCTAGCCTGCCCTCTGTGGGCAAAGCATGAACTGGCAC

This mutation is a 2325 bp deletion beginning at Chromosome 7 position 19,522,850 bp and ending after 19,525,174 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46192 GCT ATC TGA CCC ACC TAC TTC C Wild type Forward A
46193 GCG TCT GCC CTT CAT CAT C Common A
46194 GGC CCT GTC TTT CTT GTG AC Mutant Forward A
46195 Fluorophore-1 CAG GGA GCC AGG CCT CT Quencher-1 WT Probe
46196 Fluorophore-2 CCT CCC TCC CAA GGT GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46192 0.40 uM
46193 0.40 uM
46194 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.