Protocol 31602: Probe Assay - Phldb1<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=  115 bp

Wild Type = 137 bp

>chr9:44715014-44715150 137bp GAGAAACCGCCTTTGAAATC AAACGAAAACACCGAAGAGC

Sequence

Wt Sequence (deleted region in lower case):

 

TTGCTCTTCCTAAAAGAGAGGCACAGAAAGTTGGGAAAACCAAGGCAAGGAGGACCATCTTGCTGGAGCGAGTTAGAAATGACTAGACCTAGAAGTACGTACCTGGTACTGTTGCTGACTCTGTCCTGAGTCATTCAGAGTTAGAGCCAGGGAGGCCTTGCCTACCATTTCCCTTTATAGAAGGGGAACCCAGGGCAAGAGAGTTTGAATAAAgctgtagaacaggtaggtcagtggcagagtgggttgaactcagctctcctggctccttctccagagccctcgaggccccgttgccctaggatgaggtaggatggtactttcttgcttgtgcatgtatatggctccctggagccaaaaactggagcttgaggcctctttgcgtttctccctcagctgaatcggagagcctggtcaatgggaaccacacagcacaacctgcaacccgggcaccgtcagcctgtgccagccacagttccctggtgagctctattgaaaaggacctgcaggaaatcatggattcattggtgctagaggagcctggagctgcaggcaagaagcctgctgccacatccccattgtcgccaatggctaacggcgggcgctacctgttgtcccctccaaccagccccggtgccatgtctgtgggctccagctatgagaacacctctccagccttctctccactttcctcaccagccagcagcggaagctgtgctagccattcaccaagtgggcaggagccaggaccctctgtgcccccattggtgcctgcccgctcctctagctaccatttggctctgcaacctccacagtctcgcccaagtggttcccgctcctctgacagtccccggctgggcagaaaagggggtcatgagagacctcccagccctggtctccggggtcttctgacggacagtcctgccgctaccgtcttggctgaggcccgcagaaccactgagagtccccggctaggtgggcagttacctgtggtggctatcagcctgagtgaatacccatcttccggtgcccgcagccaacccgccagtattcctggcagccccaagttccagtctccagtgcctgctccccgaaacaagatcggcacactccaggaccgccctcccagtcctttccgtgagcccccaggcacagagcgggtactaacaagcagcccctctcgacaactggtgggcagaacattttcggatgggttagcagcaactcgtaccctccagccgcctgagagtccccgcctgggccggaggggcctggacagtatgcgggaactgcccccgttgagtccatctctgtctcgaagggctctgtccccactgccagcccggactgccccagatcccaagctaagcagggaagtggccgagagtccccggccccggcgctgggcagcccacgggacttcacctgaggacttctccctgactctaggagcacggggtcgcaggactcggagtccctcgcccaccctcggggagtccctggcacctcgcaagggtagcttcagtggcaggctgagcccagcttacagtctgggctctcttactggggcttcacctcgtcagagcccccgtgctcagcggaagctctccagtggggacttgcgggtacccattccaagggagaggaaaaacagcatcacggagatcagcgacaatgaggacgaacttctggagtaccaccgtcggcagcgccaagagcggctccgggagcaggagatggagaggctggtgagccaatgccagggaggctgccgctgcccaaggcggtgcctctgccagggtgtgggcaggccagccggcagaacaggaggggccatgtacagggcctgggcggattcaggatgaaccatcaggctaagtgacctgtgtctcaccatcctgataccctggagagaaaccgcctttgaaatcactaaggctttctaaattttttagttttgtatacgtgtgtgcaggtgtgtgtctgtgtgtgtagGCCAGAGATGAGTGGGTAGTGTCTTCCTCAATTGCTCTTCGGTGTTTTCGTTTGTTTGTTTTGTTTTTTTGGGGGGGGGGAGCGTTAAGACAGGGTTTCTCTGTGTAGCCTTGGCTGTCCTGGAACTCATTCTGTAGACCAGGCAGGCTGGC

1782 bp deletion beginning at Chromosome 9 negative strand position 44,716,848 bp and ending after 44,715,067 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37251 GAG GCC TTG CCT ACC ATT TC Mutant Forward A
37252 AAA CGA AAA CAC CGA AGA GC Common A
37253 GAG AAA CCG CCT TTG AAA TC Wild type Forward A
37254 Fluorophore-1 CCA GGG CAA GAG AGT TTG AA Quencher-1 MUT Probe
37255 Fluorophore-2 TTT GTA TAC GTG TGT GCA GGT GT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37251 0.40 uM
37252 0.40 uM
37253 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.