Protocol 37473: Probe Assay - Pgap1<em1(IMPC)J>Alternate 2
Version 3.0

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

>chr1:54550979-54551108 130bp TGACAAAGCGTTATCCAGCA TGCTTATAGCTTCCAGCATTACC

Mut = 142 bp

Wt = 130 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

CAGTGACAGATAGCCATTGTCAGGGATTATTTTAACTGATTGGTTAGAATTTCAACCATACGTATTTTATGCTTTTATGAAGGTTATACTCTTCAAGATCCATATAATTTAAGAATAATCCCctgcgtgaaaaacgataacttgttttgtgcattgggtatggtttatagcattcataatatttgtatactgtaccattttaaagttctttatgtttgcattgagtctgagttgtattaataaaagtgattaaaatggacccatttatttatttatttatttatttattttctgtagaaaatagagctcccaaagaaactgacaaagcgttatccagcatatgagttatatctttacggagaaggatcgtacgctgaagaacacaaaattctccctttgacaggaattcctgttctctttcttcctggtaatgctggaagctataagcaaggtaggtagtggaagaaagacgttaaagctgtgagactcagctccagacttgcaatcatgtgcgtaatgccagagtcaagtgctgtaagtgaaaagcttactctgtcaggtactgtacatgccacttcccaaaagaaagcaaacatttaagatagaaggaaaagaagagtcgaacactacttcgcagagagtgagaagaatgagatggtgtaggtgggcgaatagattatacaggcattcagaaatcagatggcatagggtagggggaatagattatagaggtactcAGAAGTAGAGGAAAGGTTAATGTTGGAAATGAAGTTTCTGAGAATGGAATCTGCAACATTCAGCGGGGAGAGAAATCTTAAATTATGTTGAACAAGGAAGAGATC

This mutation is a 615 bp deletion beginning at Chromosome 1 position 54,550,691 bp and ending after 54,551,305 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51391 TGA CAA AGC GTT ATC CAG CA Wild type Forward A
51392 TGC TTA TAG CTT CCA GCA TTA CC Wild type Reverse A
51393 AGC CAT TGT CAG GGA TTA TTT TAA C Mutant Forward A
51394 CAT TTC CAA CAT TAA CCT TTC C Mutant Reverse A
51395 Fluorophore-1 ACA AAA TTC TCC CTT TGA CAG GA Quencher-1 WT Probe
51396 Fluorophore-2 ACC ATA CGT ATT TTA TGC TTT TAT GAA GG Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51391 0.40 uM
51392 0.40 uM
51393 0.40 uM
51394 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.