Protocol 36569: Probe Assay - Plbd2<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

>chr5:120499165-120499262 98bp GGAACAAAACTAGGGCTGGA CTGTGGGCAGACTCAGGTGT

Mut = 111 bp

Wt = 98 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 AGGTCGGGAGAGGAGGCTGGTTTAGTCTTTGTTCCTCTGCCCCAGCACTTCCTGTCTCAGGTATGTAGCCCAGCAGGCATGTAGGTACAGACATGCAGGCTAACATCTGTATGGGCAGCTGCTGCTCACCAGGTGCAGACAGGCAGGCCAACATCTGTATGGGTGGCGGCTGCTCACCAGGCTGTTTTCATGGTCTAAAACTAGAGACTAGGCAGGGATGGTGTCACACATCTGTGATCTCAGCACGTGGAAAGCTGAGGCAGGAGGATTGGAGTCCCAGGCCAGCCTGGGCTACAGAGTGAGACCATGTTTGAAAAAGGGACAGGAACAAAACTAGGGCTGGAGCAGttgcctgccacacgagggcctgaactccatgtgtagtgtaaggaataaaaagccacacctgagtctgcccacagccgtgggtctggctgtgtggctgtcttttcaggggtccctgctgctaactgactgtccttcttctcccctccaggtgggcctatctggacctgagcacaaatggcaggtacaatgacagcctgcaggcctatgcagctggtgtggtggaggcctctgtgtctgaggaggtaaggccagtgtgggagtccagggtcccagctgcctgcccctggccaagcccagacatcctgagggccgtcagtggggttgATGCATGTGTTGAGAAAGGGTCCCCATCTCCTGTCCTCCATTAGGGGAGAAAAAGAGCCCTGTTCTTCAAAGACAGCACATAGGCTGGACCTGGTGAAACACACCCACAATTGCATGACTTGGGAGGCTGAGACAGACTGCTGAGTTCAGGGGTAGCCTGGGTTAGAGACAAGAAACAACAAAAACTTCCAGGCTATGATTCATGCCTGTTGTCCTAGCACTGGGGGATGGAGGGAAGACAAGGGTCAGGAGATCAGAACCAGCTGTGACTACTCCCC

This mutation is a 324 bp deletion beginning at Chromosome 5 position 120,498,915 bp and ending after 120,499,238 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49129 GGA ACA AAA CTA GGG CTG GA Common A
49130 CTG TGG GCA GAC TCA GGT GT Wild type Reverse A
49131 CAG CCT ATG TGC TGT CTT TGA Mutant Reverse A
49132 Fluorophore-1 CCA CAC GAG GGC CTG AAC T Quencher-1 WT Probe
49133 Fluorophore-2 AGA TGC ATG TGT TGA GAA AGG GTC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49129 0.40 uM
49130 0.40 uM
49131 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.