Protocol 32542: Probe Assay - Hnrnph2<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

>chrX:134605508+134605607 100bp CTCATGACTATGCAGCGC CCCGCCTCATTCTTTCAAAC

Mut= 95 bp

Wt= 100 bp

Fam=Mut

Hex=Wt

X-Linked

Sequence

Wt Sequence (deletion in lower case):

 

ATATGGCTCCTTTGAAAAACTTACTGATGTGAACTTGTAAAATAATAGTTTTCTTTGTTTCTCTTACTATATTATATACACTTTTTCATGACAATAGTTTTCTAATTTTTTTAAAGCCACATTGAATTTGCATTGAGCCAAGCTTGCCACCAAGAGTCCATCAGTCACCatgatgctgagcacagagggcagggaggggttcgtggtgaaggtcaggggcctaccctggtcctgctcagccgaggaagtgatgcgcttcttctctgattgcaaaatccaaaatggcacatcaggtgttcgtttcatctacaccagagaaggcagaccgagtggtgaagcatttgtcgaacttgaatctgaagatgaagtgaaattggctttaaagaaagacagagaaaccatgggacacagatatgttgaagtattcaaatccaacagtgttgaaatggattgggtgttaaagcatacaggtccaaatagtcctgatactgccaatgatggttttgtacgactcagaggactcccatttggctgtagcaaggaagaaattgttcagttcttttcagggttggaaattgtgccaaatgggatgacactgccagtggactttcaggggcggagcacaggggaagcttttgtgcagtttgcttcacaggagatagctgagaaggccttaaagaaacacaaggaaagaatagggcataggtacatcgaaatcttcaagagtagccgagctgaagtccgaacccactatgatccacctagaaagctcatgactatgcagcgcccgggtccttacgataggccaggggctggaagagggtataatagcattggcagaggagccgggtttgaaagaatgaggcggggtgcctatggtggagggtatggaggctatgatgactatggaggttataatgacgggtatggttttgggtctgatagatttggaagagacctaaactactgtttctcaggaatgtctgatcatagatatggagatggtgggtccagtttccagagcaccacggggcactgtgtacatatgagggggttaccatacagagccaccgagaatgatatttacaattttttctcacctcttaatcccatgagagtgcacattgaaataggacctgatggaagagttactggtgaggcagatgttgagtttgctactcatgaagacgctgtggcagctatggcaaaagataaagcaaatatgcaacacagatatgtggagctcttcttgaattctaccgcagggacaagtggaggagcttatgatcatagctatgtagagctctttttgaattctacagcaggggcaagtggtggtgcttacggtagccagatgatgggagggatgggtttatccaaccagtctagttatggaggtccggccagccagcagctgagtggtggctatggagggggttatggtggtcagagcagtatgagtggatatgaccaagttcttcaggaaaactcaagtgactatcagtcaaaccttgcttaggaagcaagggaccatccaataacatctacatgaataaaagctatctttataggaactggatagagtagggagggtgtctttatatccagtatgattggtaaATGGGAAGTACAACTGCTTCTGATCACTATTGGTCAGCTTCTTTCTTCTTTTTCCTTTTTTTTTTTTTAAAAAAAAGAACAAAAATTTAAGTTTTAACAGTTTCGCATTACAAGCTTGTGATTCATGCTTACTGTAAAGTGAAAGTTAAGATTGTTTTCAAACTTTAAGCTCAGCAATTTTGAACACTGAAAATATTCATCTAGGATGTAATAACAAGTTCAGTATTG

 

This mutation is a 1451 bp deletion beginning at Chromosome X position 134,604,908 bp and ending after 134,606,358 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39357 CTC ATG ACT ATG CAG CGC Wild type Forward A
39358 CCC GCC TCA TTC TTT CAA AC Wild type Reverse A
39359 TGA ATT TGC ATT GAG CCA AG Mutant Forward A
39360 GAA GAA AGA AGC TGA CCA ATA GTG A Mutant Reverse A
39361 Fluorophore-1 AGA GGG TAT AAT AGC ATT GGC AGA Quencher-1 WT Probe
39362 Fluorophore-2 ATC AGT CAC CAT GGG AAG TAC AA Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39357 0.40 uM
39358 0.40 uM
39359 0.40 uM
39360 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.