Protocol 37976: Probe Assay - Cfdp1<em1(IMPC)J>-Alternate3
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

>chr8:111841642-111841778 137bp GCTTAGAGTTTGGTGGGATAAAC TCCCTTAGTGAAGACATCTAGTTTG

 

Mutant=  147 bp

Wild Type = 137 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletions in lower case):
GAACACAGACATACACATAAATAAATGAATAAATATCTCTAAAAAAATTAAATCTCAGTTTACCGTCTCCATCTCTCTTACTGTGTTATTAGTAATCCCAGATGTAAACTTTGAGGTGTGTAAGGTGatccatggattggggagaggcagtgagtgacttgcattctgtcttgtcttccttttcctgggtggctgttggtgagtaggaaaaggaaacaaagtggcctcttgttagaagaagaggaagatggcaaggaagactctggaggtagtagcagtgaagaagatgaagaggagcaagaaggaggccttgggtcagagaatgcaaggaagaagaaggaagatgagttgtgggccagcttccttaatgatgtaggaccaaagtcaaaagcagccccaggttcacaaactaaggtaagcttagagtttggtgggataaacacctttccagaaacgccctcacttccctgggagcTTCCTATTGTTTTAATTCGCAAACTGACTTGAAATTTGAATCTCAAATCAAAAAGCAAACTAGATGTCTTCACTAAGGGAAAAGCAAAGCATTGCTTTCTTTCTTTC

 

This is a 348 bp deletion beginning at Chromosome 8 position 111,841,722 bp and ending after 111,842,069 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
52434 GCT TAG AGT TTG GTG GGA TAA AC Wild type Forward A
52435 CCG TCT CCA TCT CTC TTA CTG T Mutant Forward A
52437 Fluorophore-1 CTT TGA GGT GTG TAA GGT GGA TTC C Quencher-1 MUT Probe
52674 TCC CTT AGT GAA GAC ATC TAG TTT G Common A
52677 Fluorophore-2 TTT CCA GAA ACG CCC TCA CT Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
52434 0.40 uM
52435 0.40 uM
52674 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.