Protocol 36571: Probe Assay - Plekhg2<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

>chr7:28368736-28368836 101bp GTGCTTCGTGCAAAGAGTGA ATGGGAGTCAGCACCACCT

Mut = 100 bp

Wt = 101 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 GGCATTACCAACACCCCCTTTCCCAGGGCTGTGGTGTCACTCCCTAGGTCACGACATGCCAGCCATGCCAGGGTTATCAGGCTGAATAGTACGAAGCCGTGAGGAGGCAGAATTTCTGGGGTAGCCATCTGAGGCTCCTTCCTACCTGTCTGTTGCCCTCTggagtcttgtcatgggacaggggggcaggcaagccctttaccttcccctttgcccgcccacagtgagctcctggaggacttggaaggctgcagcagtgcagggggcatcgccgagtgcttcgtgcaaagagtgagtaggacagcttgaggctgggggacggacgctgggccgggattcatggggccatggggatctaggtggtgctgactcccatgtccccacagagcgaagattttgacatctatacattgtactgcatgaactacccaaggtgaggcaaagggagtccctgtttggcccccgggtattcccacgagccaatgtgccaaaccactaccccctaccctttaccgcccccaaccctgtgcccaggggcagccccttctagaccttgatagtcactggcctccttaactaccctgacctgatcatccctgaccctggccacctcctcccagccctgcctgctggagtgccagccaccactgatctatgctgtgccccccagctccctggccctgctccgagagctgtcagtgtccccgccagccactctgtggctacaggagcgtcaggcccagctccgtcactccctgcctctgcagagcttcttgctgaagcctgttcagcgcatcctgaagtaccatctcctgctgcaggtcagctgtgcccctgaggcctggcttgtggagagggggcatggttgggtctaacagccacccctggtaccctgactgtctgtggtctgcccctcccctttcatgttgctctgcctcctgtctcactcctcttttctctttttctcccccatccttctaccctgctggatgactgtcaggaactgggcaagcactgggcagagggcccagacagtggagggcgagagatggtggaggaagccattgtatccatgactgcagtggcctggtacatcaatgacatgaagcgcaagcaagagcatgctgcacgcctgcaggtggcccccggggtgggctggggcacctgagctgggggatgttgctcagtgtgtcccggtcactaacgcctctgttttggcttgtatatgtgtgccaccaacctgggcctgtctctatgcctgatgtggtcccagcaggaagtgcagcggaggctgggtggctggactggcccggagctcagcgcctttggagagctagtactggaaggcactttccgagggggaggcgggggaggccccaggcttcgtgggggtgagcgcctcctcttcttgttctcccggatgctgctggttgccaagcgtcgggggcctgaatacacctacaagggccacatctttgtgagtatagagtggggtctggctcagagagtcagaactaatgtatgttcccctgagttcccgaaaggggacagactggcttccaaggaccatatttagctgcgcccagtgacatttgacctttgattccagttactgggaaggctgagacaagaccaaaagttcaagactaacctgggctacagaaaaggttcaagaccagtcccgggcagcctgattaaaacaacattaacggctagaagtagGGCATGGTAGCTCACTGGGGAGGCAGGGGGGTTGAAGTAAAAGCCTGAGGCCAGTAAGACCGACAGGTGAGCTCAAGGCCAGCGTAGGTTATGTAGTGAGATCCTGTCTCAGGAGGGCACACTGATCTCCAGGTTCTAGGGTAACTAGGACTACAAAGAGAGCCAGACCCTGTCTCAAAACCCAGCCAAAAAAGCAACAGTGAGCGTAGCTGGTGGCCACAGGAACCAGTGTGTGAAGATGCCACAAACTTGTCCGCTGACCTCCACTGGCC

This mutation is a 1561 bp deletion beginning at Chromosome 7 position 28,367,390 bp and ending after 28,368,950 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49136 GTG CTT CGT GCA AAG AGT GA Wild type Forward A
49137 ATG GGA GTC AGC ACC ACC T Wild type Reverse A
49138 GGC TCC TTC CTA CCT GTC TG Mutant Forward A
49139 CTC ACC TGT CGG TCT TAC TGG Mutant Reverse A
49140 Fluorophore-1 TGA GGC TGG GGG ACG G Quencher-1 WT Probe
49141 Fluorophore-2 CCT CTG GCA TGG TAG CTC ACT G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49136 0.40 uM
49137 0.40 uM
49138 0.40 uM
49139 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.