Protocol 46831: Probe Assay - Rnase4<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

Mutant=  63bp

Wild Type = 124 bp

>chr14:51106133+51106256 124bp CTGTATGTTGAGCCATCTGT AATGGCCATTTGTATTTGAC

Sequence

Wt Sequence (deletion in lower case):

TTTTCTTACAAAACCCAAATTCCAGTTTTATGTAGATTTATCTTTCCTTGTTTTATACTCCAGTCAGGCAGGCCTGGGCAACTGACCACCCAGTCATGTTTGTTCTGACTTGTTCTGTTAGCTGTACGCCTGTCAATTAGTAAAGAGAACAGTGTTTGCTTTCAAGGAAGGCCATGTTGACAAACTAGCCACTCTCAGAGaaaggggtacaaaagggaagagcaggaagaagggggagatcttaagcagaaggaggggcaagagagggttctaagagtttagtttgcttttctcattacctctccagagcccagtccttaccatcctttctccttgttttcttctcaggcactttctaggtaatgatggatctacagaggactcagtccttgcttctgctcttggtgctgaccctgctggggttagggcttgtacagccctcctatggccaggatcgaatgtaccaacggttccttcgacagcatgtggaccctcaggtgacaggtggcaatgacaactactgcaacgtgatgatgcagagacggaagatgacttctgtccagtgcaaacgcttcaacaccttcatccacgaagacatctggaacattcgtggcatctgcagtaccaccaatatcctgtgcaagaacggccagatgaactgtcacgaaggtgtagtgaaggtcacggactgcagagagacagggaactccaaggcccccaactgtagatacagggcaagaaccagcactaggcgagttgtcattgcctgtgagggtgacccagaggtcccagtgcactttgacagatagatgctgtcctgcagctgttacagctgatagttgacctctgagtcagtgagaccaacaatgagtggtggccccaggctttgtttcctttgctcgtgccttatactcacatatatccaatataacccatggtgttaagcgcatcccatccaagggaagaaagaacccactgctcacagcactgatggcttggttctcccagattctatcccctggaacttatgcattacatgtattcagatagtatctcaacgattatagagagccttttctgtccaagctgtctcattccagtcctctaacaccccttgagaccaattctgccactgtatttagaagctttgagttaaaggatttgacgaccacaaaaaaccaaaaccaaaaccaaaaacaaaaacccagtggaagatctcagacaaaaatccaactggacgggctactcacaaaaggtgttacccacttcgttatatatactttctgttattccaaaatgtcatcattagggaagccactaagagtttggaaataggggctggagagatgcctctgtggttaagagcattggctgctcctctagaggacccaggttcaattcccagcgcccacacatcagctcacaactgtctgtaacccagttctaggtgatttgataccctcacacagacatacatgtagacagatcaccaatgcatataaaattaaaataaagattttggaaacaacatattgccaaaatgagaattacttggtattattttggtctgtggcttctaagattttttaaaatacatgaaaaatgtagtaaatcactaaaattcatttatccttctcctttgaaagattttcaatattttagtataataaagagctgtatgttgagccatctgtgtcgggatttgtgttctcctgttctttttcagttcctctctagtctatgaactctcatgtcccttagtgtccctatACATTCTGGTCAAATACAAATGGCCATTAGATCCATCATGTGACATCTGACTTTGTCTCCTTTACTCCACTGAAAGTGATCCCTCTCATACACAAGACCTGAAATAAGAACTCAGAAGTGGCCTTTGCTAAAAAGCACCAATTCTTTATGCAACAAATTGAGTGTGTTGCATGATTAAGC

 

This mutation is a 1570 bp deletion beginning at Chromosome 14 position 51,104,659 bp and ending after 51,106,228 bp (GRCm38/mm10)

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
68976 CTG TAT GTT GAG CCA TCT GT Wild type Forward A
68977 AAT GGC CAT TTG TAT TTG AC Common A
68978 GGA AGG CCA TGT TGA CAA A Mutant Forward A
68979 Fluorophore-1 CAT GTC CCT TAG TGT CCC TA Quencher-1 WT Probe
68980 Fluorophore-2 CTA GCC ACT CTC AGA GAC ATT CT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
68976 0.40 uM
68977 0.40 uM
68978 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.