Protocol 35137: Probe Assay - Ccdc51<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

>chr9:109092669+109092776 108bp CTCTGCTTGTTGGGCCTTC TCTCTTCCTTTCAGCACCAGA

Mut= 105 bp

Wt= 108 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 GCTGCTTATTTTCCTATAGACGTGCTCTGCATGACTGCCCGGGGGAAAGAGCACATGGCTACCACACACACACACACACACACACACACACACACACACACACACACCGCTCTGCTCCCACACCAACAGGTATATTCTGTCTTCATGCAGCAGGTGTTTGTAGAGCCTgtggcaggggagtgtcctctgcccccgtggctgtagtcacaaggctccttgccgatgatggtctgataccactttgttcccacaggaagagaagaggctccgaatagcatacctgcgtgcagaagactcagagcgagagaagttctctctcttctctgcagctgtgcgggagagtcatgagaaagagcgtacaagggctgagaggaccaagaactggtcccttattgggtcagttctaggagctctgataggtgtggccggttccacctatgttaaccgcgtccggctacaggagctgaaggccttactcctggaagcacagaaaggtcctgcgagtctccaggaggccatccgtgaacaggcttctagctattccctccaacagaaagacctccaggaccttatgatggatctgaggggcctggtgcacgccgagcagggccagggctccgggtcaccaacaggttcttctaccagaggaaaagacatagatggcctttcagccaccatgaaagagcagctccgtcattccaggcaggtctattcctgcctagagggtttacgagagcagcttgatggcctggaaaagacttgcagtcaaatggctggggtgcttcagctggcacaggctccagcacatccaggcacggtggggccggtggatggggccctgcccagctccttgctggaacacgggagtgtgatcttggccctgtcagagatggagcagaggctagaagcccaggctaacaggaacactgtctctagcacactggtcacctgtgtgaccttcctggccacattgcctctgctctacatgctgttcaagaccagttaaccctggggaccttcttcaaagggaccgaagggtgactgtcgcttttgttggtgatataatgaatcttgaggtacacacaacctcagcctgactctgaatgccatctgaggggctcaccatctgagatatattttattttgtaggtagtacctatttacattaattatcgaagttttgtgcaaatgtccaattaaaatatctaagaatttgtattatttaaaaggtagttatatcagactcagttttatcattgatttcagaagtctgggacttgaagtttagcctggagtgggggctcaccccctttagttcctttctcatctcctagtggtcctagctcaggactatgttggatgaagttagccctggattctcacttagaatgtcctgacactgttgccctctgcttgttgggccttcctgtgtgtgggctcaatcgagtagagcctcacctggtgcGTGGCTTGGGGAAGAAGAAGCGATGCCGTTCTGGTGCTGAAAGGAAGAGACTGGGGTCATCATTACAGATCCGTGCTGGTCTGCATATCCCTGATACAGAGTCCTGTCCATGCAACCTGGGACCCTCAAAACGGCACAGGCATAGTCCAGGCCCTCGCTGGCAGCAGTG

This mutation is a 1297 bp deletion beginning at Chromosome 9 position 109,091,430 bp and ending after 109,092,726 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
45352 CTC TGC TTG TTG GGC CTT C Wild type Forward A
45353 TCT CTT CCT TTC AGC ACC AGA Common A
45354 GCT CCC ACA CCA ACA GGT AT Mutant Forward A
45355 Fluorophore-1 CTC AAT CGA GTA GAG CCT CAC C Quencher-1 WT Probe
45356 Fluorophore-2 TGT AGA GCC TGT GGC TTG G Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
45352 0.40 uM
45353 0.40 uM
45354 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.