Protocol 37690: Probe Assay - Bcl7c<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

>chr7:127707637-127707737 101bp AGCGCACTTACACACACACA AAGGTGCTGCATCAGGTTG

Mut = 100 bp

Wt = 101 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

 CCAGAGGTCTACAAAGCGTGGTCCGGAGTGGTGCTTTCCCTGAGCGGGAGCCCTCATCTATGCCCCGccccctgaagcttttgacactctagcaagcttggccccgggtcttcattgccagagtccctggccaggccttggtggcgccctgctgagtgccccaggtgtctgcaaaaccgcagctccaagttatatccgtttctgctttgccctggggtttgagggctctttctgcttctgtgccagtgttagcgaagctgtccctaccctagcacacctccctgacctccagtctgtggggctcatccttgggcctccctgatccagtctcctgacctcccacagggagaagcgctgggtgactgtgggagacacttcccttcgaatcttcaagtgggtgcctgtggtggatccccaggaggaggtgagcaagccccaacccccaacaggccttgcatctgtgcagcctcaagcaatttccctcccagccttggcctccaccttcagtcggcttggagactccagtgtggagcccaggcagatggtaacgattattagggggcgcgagcgcacttacacacacacacacacacacacacacacacacacacacactttctccaagttcaaggcctaactcaatctacacaacctgatgcagcacctttcgcttctctgagcctaggtttccttattcgtattgaagggctttgtaagagctccctagacaactagctctgatcttacctctctaacccccaggagaggcggcgggcaggaggcggggcagagagatcccgtggccgggagagacgtggtaggggcaccagtcccagagggggaggccccctcatcctactggatctcaatggtatgtagagattctggaatggagttgggtgggggggactgaaggaatcccctggggatctctggctaattaacgcgcggcttttgcttcatcagatgagaacagcaaccagagtttccattctgaaggttcattgcaaaagggtgctgagcccagccctggggggacgccccagcccagccgccctggatcaccaactggacccccagaagtgattactgaagatactcagcccccacaattgggtcaggagagaggtagggccctgtggctctcaggagccctctcatcaaagttgaacccaccatcctgtccttgtttcttatattatgtcctctcacccggtcccgcctccctcagggaatcttccctgaccctggcagagatcacctccagctgtgcctcaatcctccgGCTTCTTAGATCTGTAGGGAGCATATCACCAGCTGACTTTTTTTTTTTTTTTTTGCAGATTTATTTCTTTGCTTGCTTATTGTCTTTTTCTGTTATCA

This mutation is a 1218 bp deletion beginning at Chromosome 7 position 127,707,019 bp and ending after 127,708,236 bp (GRCm38/mm10). There is a 26 bp insertion (GGGCCAAGCTTGCTAGAGTGTCAAAA) at the deletion site. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
49916 GGA GTG GTG CTT TCC CTG AG Mutant Forward A
49918 TGG TGA TAT GCT CCC TAC AGA Mutant Reverse A
51664 Fluorophore-1 CGG GGC CAA GCT TGC TA Quencher-1 MUT Probe
51946 AGC GCA CTT ACA CAC ACA CA Wild type Forward A
51947 AAG GTG CTG CAT CAG GTT G Wild type Reverse A
51948 Fluorophore-2 ACA CTT TCT CCA AGT TCA AGG CC Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
49916 0.40 uM
49918 0.40 uM
51946 0.40 uM
51947 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.