Protocol 34356: Probe Assay - Krt40<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

>chr11:99540354-99540473 120bp ATCTCAGGGAGCCCACAAC TCTCTTGGTTAGGTCTCTTCTCA

Mut= 117 bp

Wt= 120 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GACATTTAAACCTAATGGAAAGAGGGGATGGGAAAATGACTCAGCAATTAAGAGTGCCGATTGTTCTTCCAAAGGACCCAAGTTCAGTTTCCCGTACCCATCTCAGGGAGCCCACAACTGTCCATAACTgtagctccgtgtggcatacaggcacacttaaatgtaaatttttaaaatctttcttaaacagaagtaatgagaagagacctaaccaagagaatctgaacttttacccagtgtgccaaaaatcaaagtgcaatgcttttttcatcacattaacatcacagacacacacacacagagcactctgacattcttcactccatttccacccacaggaggtcaacctgcttcgtgagcagcttggcgatcggctcagcgtggagctggacactgcccccactgtggacctcaacaaggtcctggatgagatgcgatgtcaatacgagagggtgctggccaacaaccgcagggatgcagaggagtggttcgctgctcaggtcagtgccctcggtaaaggactggcttccgggagggtgccctgcctgagctctctaatggggcctttgtttcagacagaagagctgaaccagcagcagaagtccagcgcggagcagttggagggctgccagactgagatgttggagttgaagcgcaaggccaacactctggaaattgagctccaggcgcagcaaaccctggtgcgtgggatgaacagtgccagcctttgtcagcagcatccagagatgctatgctctttgacagaagtccagggagatgctcacagttcagcagcaggtctgggtatccaattagcctagctggcatttccctccctttcttcacatatgttacttgtccacagtagaaacagaatgacagatcaacgggctttgtcctctcaccagggtgcctccctggccctcctaagcctaagcatgtctccaataccatcagctagagacaagaaaagtctcagaacacagtgggacagggagagctgagccaaaggcccttaagcttagcaacagattttcctttcctctaatcgatgaacagttttacagattcaccgtagtgtctaaaacaaaaaaacaaacaaacaaacaaaaaaaaaccacatcaagtttcaagtggtcatacaacagctaagacccgcacatcaccatagccctccccacccacacaagaacaccatgcagtgaaagtcacgtataccaggtcccatcaggcactgtggtaccagttgagcttcatccaaggagtttacctagatgaacgtcacctgagcatccacctaggagaggcatggtgcaatctatggcagacaccttcactagcccgggtgaacaggtgggggaaagctagggtcaaaggaagaaagttccagaatgtgctgcagttctcagtagaaacagaatcctcctggtggttgaaccctgatcttctttgtaccaacactcactgcgcagatgctcagactcaagggtttcttcacaaatgatgacaaccctccttgagctacccaaaaaaagttctggctgacaattgttctaatgatagaagcgacacatttaaaggccttgagttgcttctaggcactgttccaaattctccccaagtatcagctcatctcacctccccagcctcccccatgacataggtactttgcattctccattgactctagaataaaacaggctcagagaagctaagtaactggccctgaggtctctgagctagaaaggggtacatcgagggtctgtcatagggtccttctgagaaggttctactcttgcagacggagtctctggagtgtactgtggcagaaaccgaggcccagtacagcacacaactggcccaaatgcagtgcctaattgacagtgtggagcaccagctggctgagatccgctgtgacctggagcgtcagaaccaggagtaccaggtgctgctggacaccaaggcccggctggagtgtgagatcaacacgtacaggggcctgctggagaaggaggacagcaggcaagttacacagacccccagagtccccccaggagaaccctgtttcctcaaggcctgactaaggccaattccatgtttcggggtcttccttttcaaacagtgaaagaaaggcgtggccagGTAGCTCAGTGGGTATGTCAGTTACTTCTTTGCTTCTGTAAACATGGCCAAAGACAACTTGAGGAAGAGAGAGTTTTGCAGATGGGTAAGAGTATGTCATGGCTGCAAGCATCAGGCCTGACAGCAGGAAGT

This mutation is a 2013 bp deletion beginning at Chromosome 11 position 99,538,431 bp and ending after 99,540,443 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43401 ATC TCA GGG AGC CCA CAA C Common A
43402 TCT CTT GGT TAG GTC TCT TCT CA Wild type Reverse A
43403 ACC CAT CTG CAA AAC TCT CTC Mutant Reverse A
43404 Fluorophore-1 AGC TCC GTG TGG CAT ACA G Quencher-1 WT Probe
43405 Fluorophore-2 CTG TAG CTC AGT GGG TAT GTC AGT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
43401 0.40 uM
43402 0.40 uM
43403 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles (Touchdown)
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

This is the only strain that uses this protocol.