Protocol 35472: Probe Assay - Rrbp1<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

>chr2:143988068-143988162 95bp AAAAAGGAGAGCCTGGTGAG CAAACAGCAGAGCACTAAGGA

Mut= 112 bp

Wt= 95 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case; and insertion with carrots ^g^ (TTAGTGCTTAG insertion):

 TGCTTAGCTTTGCTGTCTTTGTCATGTTGCCACTACTCTCTGATGTTTCACTTCCGGACAGCACTTGTGCTCATGAGTGCTTTAAATGGGGCGGGTTCAAGGGGGCACCTTGAACTTGGGCCATGGCCATGGGCTTTGGAGGCTACAGAACATTAAAAACTTGAGTCGTTCCC^ctgtacttctccatccgcctgtttgtatctccagtactaaaagaactattgataccgtaggtaaaaattacccatttctgaagagagggtaagaaagaggagcaaacatggctttgctcctactctgaatttagtctgaagacagtgagaaaccttgagtttggtgataggggatggaaactgtccagtccctcagtttccccagaagtaatctacttgtctgtgttggcaggtggaaagcaagccagccaagatggatatctacgacactcagaccttgggggttgtggtctttggcgggtttatggttgtttctgccatcggcatcttcctggtgtcaaccttctccatgaaggagacgtcgtatgaagaagcccttgccaaccagcgcaaggagatggcgaaaactcaccaccagaaaggagagaagaaaaagaaggagaaaaccgtggagaagaaaggaaagaccaagaaaaaggaagagaaacctaatgggaagatacctgaacatgacctggatcccaacgtgaccatcatcctcaaggaaccagtgcgtgtatccgctgtggctgtggctccaacttcagtccattcttctgtgggccatacccctatagccacagtaccagccatgcctcaggaaaagctggcttcctcccctaaggacagaaagaagaaggagaaaaaagtggcaaaggtggagccagcagtcagttctattgtgaattccatccaggttcttgcttcaaagtctgccatcttggaagccacacccaaggaggtgcctatggtggctgtgcctccagtgggctctaaggccagttctcctgccaccagcagtcagggcaagaagggacagggagcccagaaccaggccaagaagggggagggagcccagaaccaaggcaagaagggagagggggcccagaaccaagctaagaaaggagagggggcccagaaccaggccaagaagggggagggggcccagaaccaaggcaagaagggggagggagcccagaaccaggccaagaagggggaaggaggccagaaccaggccaagaagggggagggggcccagaaccaaggcaagaagggagagggggcccagaaccaaggcaagaagggggagggagcccagaaccaggctaagaagggggagggggctcagaaccaagctaagaagggtgagggggcccagaaccaaggcaagaagggagagggggcccagaaccagtccaagaaaggagagggggctcagaaccaggccaagaagggggaagggggccaaaaccaggccaagaagggagagggggctcagaaccaggccaagaagggggagggagcccagaaccaagctaagaagggggagggggtccagaaccaggccaagaagggggtagaaggggcccaaaatcagggcaaaaagggggaagcaaaccaaaaccaggctaagaagggtgagggaggccagaaccaaaccaagaagggggaaggaccccagaaccaaggcaagaagggagaagcagctcaaaaacaggacaaaaaaatagaaggagcccagaatcaaggcaagaagccagagggaacctcaaaccaaggcaagaagggggagggggcccagaaccaaggcaagaagggagagggggcccagaaccaaggcaagaagggagagggggcccagaaccaaggcaagaagggagagggggcccagaaccaaggcaagaagggagagggggcccagaaccaaggcaagaagggagagggggcccaaaaccaaggcaagaagggagagggggcccagaaccaaggcaagaagggagaggggccccagaaccaggccaagaaaggagagggggcccagaaccagggcaagaaaggagagggggcccagaaccagggcaagaaaggagagggggcccagaaccagggcaaaaaggcagagggggttcaaagtcagagcaagaaaggggaagggacccagaatcagggcaaaaagggggatggaaatcccaaccaaggcaagaagggggagggggcttccaaccagaacagaaagacagatacagttgctaatcagggcacaaagcaagagggtgtctcaaaccaggtgaaaaagtcagaagggtcccctaatcaaggtaaaaaggcagagggagccccaaaccaaggcaaaaagaaagatggatcccccagccaggccaaaaaagtggatgcagctgccaatcagggtaaaaagtcagagatggctcctgcccagggtcaaaaggcaagcatggtccagagccaggaggcaccaaagcaagacgcacctgcaaagaagaagtctggttccaggaaaaaaggagagcctggtgagtgcccgtctgctctgtgagcttgagggaaagccgctgactgtgactga^TGGACTTCCTTAGTGCTCTGCTGTTTGCCATTGTCTTTTCACCCCAGATAGGGTATTGATGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGATAGAGAAATAACTCCACCTAGTCTCTCTTGGACTATTGAGCTTTTGGGATTTTATACAGGAGCA

This mutation is a 2404 bp deletion beginning at Chromosome 2 position 143,988,095 bp and ending after 143,990,498 bp (GRCm38/mm10). In addition, there is an 11 bp insertion at the deletion site (TTAGTGCTTAG).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46160 AAA AAG GAG AGC CTG GTG AG Wild type Forward A
46161 CAA ACA GCA GAG CAC TAA GGA Common A
46162 AGG GGG CAC CTT GAA CTT G Mutant Forward A
46163 Fluorophore-1 CGT CTG CTC TGT GAG CTT GA Quencher-1 WT Probe
46164 Fluorophore-2 AAC TTG AGT CGT TCC CTT AGT GC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46160 0.40 uM
46161 0.40 uM
46162 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.