Protocol 38023: Probe Assay - Cdkn2aip<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

>chr8:47710961-47711122 162bp CTCAAGAAGAAGGTGGTGGTG GGATTCAGTGCCTACTTGGA

Mut= 174 bp

Wt= 162 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

GCCTATCCTGGAACTTGCTCTATAGACCAGGCTGACCTCAACCTCAGGGGTCATGCCTGCCTCTGCTTCCTGAATGCTGGGATGAAAGGCACTTTGCTGCCACCCCACTCTCTGTGATTTTTAAATGAATGTTTTGTTGATTGTCATGTCTTTAGGACTCTTAGTAAAATACCTGTGTTTTTCTTCTTTCTAGAAGGGGTAGAGGAGCCATCcaaaaaacgagccgtagaaggaaaaaacaattcttcagttgagcgagatcatgggaaaaaatctgccaaaacggatcgttcagcagctcagcaagaaaacagttcgccgtccagggggtcgtccaccaaatccgagagtggtgggacctcagctcggagcagctccagtggctcccatcaggatagcgccacaagtgaaggagacagatctgtgtgcagccagagcagcagcaattcctctcaggtaaccgcagggtctggaaaggctttggagtcagaagctccacataaacgtggttcagcgtcatttgtttcttccctgttgaagtccagtatgaatagtcatatgacccagtccactgataacagacaacagagtgggtcacctaaaaagggtgctctggaaggttcttcaggctcagcctctcagagcagctcagagattgaggtgcccttgttgggctcctctggaagtgcagaagtagagttgccattactgtcttgtaagtctagttcagagacagcttccagtgggttaacatctaaatctagttcagaggcaaacatttcatcatcagtttcaaaaaacagttcatcgtcaggcagctctttactaaccccccagagcagttccacaaatccatcgctgctgacatccaaaagcacggctcaggtagctgcttcactgctagcgaccaaaagtggcgcttccttaggaagcgtgtctcagctagctgccaagagtggttctcagagtagcacttcacagttgccttcgaaaagtacctcccaggccagtgagagttctgtcaagtttgcttgtcggaagttaaccaatgaagacataaaacagaagcaacctttcttcaatagactgtataagacggtggcgtggaagttagtagcagttggtggcttcagccccactgtgaatcacggagagctgctgaacgcggctattgaggctctgaaagcaacactggatgtgttttttgtcccactgaaagaactggcagatctgcctcaaaataagagctctcaagaaagtattgtctgtgaattgaggtgtaagtcggtatatttgggcactggctgtggaaaaagcaaagaaaacgcaaaagccgtcgcatcgagagaagcactgaagttatttctcaagaagaaggtggtggtgaagatctgtaagaggaagtaccgaggcagcgagatcgaggatctagtactcctggatgaagaagcaagacccgtaaacttacctccagCACTGAAACATCCTCAAGAATTACTGTAACTTCTCCAAGTAGGCACTGAATCCAGTATCTGGTGTTTGAAGAGAAAAACTGGCTTTTATATAAGACACTGGCTTTCACAGATTTTATATTGCTTTTTTCCAGTTTTGCAGAAAATTTAAATTCTCGTTCTCTTCACTAAAGTAGAGCTGTAAATAATTTATGACAGTGCATATTAAAGGGTGT

This mutation is a 1262 bp deletion beginning at Chromosome 8 position 47,711,014 bp and ending after 47,712,275 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52805 CTC AAG AAG AAG GTG GTG GTG Wild type Forward A
52806 GGA TTC AGT GCC TAC TTG GA Common A
52807 CTT TGC TGC CAC CCC ACT CT Mutant Forward A
52808 Fluorophore-1 AGA GGA AGT ACC GAG GCA GCG Quencher-1 WT Probe
52809 Fluorophore-2 AAG GGG TAG AGG AGC CAT CCA C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52805 0.40 uM
52806 0.40 uM
52807 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.