Protocol 41951: Probe Assay - Banf1<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

Mutant=  133 bp

Wild Type = 151 bp

chr19:5364436-5364586 151bp GGGCTGAACACGTCTACTTCA CCACACTCTCTCCCACAATG

Sequence

Wt Sequence (deletions in lower case): GAGGGGCGATGCGCGGTGTGTCCGGGTGGCGGCGCGTGGAGGGGGCGGGGCGCTCGGAGCGGGGCGGGGCCTCAGGGCGGGGCCTGCGGGACCTGAGAGGCGTGGAAGAGGCCGGCAGCCCGAGTGAGGGCGGCGAGGCTGGCGAGAGCCGCGTGCTGTTCCTACCACGTGTTAGTTCCCAACGGGGTCAGTGTCTGTGTAGCCAGGGTCGCGGGCTGTGAGGGCTTCAGGGCGGACCTTGACTACTGAGTCTGGGAGTCGCTCTGTTGCCTTAGGTGCGCCACCAGCTAGTGCAAGCTGATATATCCAATATACCCAAAACCAGGACCCACGGAAAGTGGAAATTTCCGAATAgccggggatgggttggggggactcaagggagcagccacagtagctgtggtaggatattgcccttgggacttgtggtcttcagccctaacctacttttcttttttttttttttttttttttggcgggggggattcccaggttaagcctgatcacgatgacaacctcccaaaagcaccgagacttcgtggcagagcccatgggggaaaagccagtggggagcctggccgggattggtgacgtcctgagcaagaggctggaggaaaggggctttgacaaggtgtgggggggctgcaaatgccagggaggggtaggagcgaaggttggcaacaggtggtaccacgcagattgggagtgaggcccacacatgtttggggggtacctggggaggcatggtgcctccttcatttttgtgttttgttgagacagggcttctcagtgctaggattaaaggtgtggctcactgtcccctgcccttttgtgtgttttaaatatcacgaaaatgaaactaggcatagtggattgtgcttatgaccctaacacttgggaagttgagctaagaggattcccgtgggatttaatgccagccctgacaacaaaggaacaaaccctgtctttctttttttttttttttcgagacagggtttctctgtgtagccctggctgtcctggaactcactctgtagaccaggctggcctcgaactcagaaatccgcctgtctctgcctcccaagtgctgggattaaagtcgtgcgccaccactgcctggcaacaaaccctgtcttaatcaaaataaaaatggccttgccctctgaagtatttaccacctaaccctccctagcttcagagatctggcctcttcggagtggtatcagcgtggacagtcctgttgctttagcccatgagacacttagccacatgcccttttctattctgcaggcttatgtggtccttggccagtttctggtgctaaagaaagatgaagacctcttccgagaatggctgaaggatacatgtggtgccaatgccaagcagtcccgggactgctttgggtgccttcgagaatggtgtgatgccttcttgtagtgttctctggggatccctcataccccagccacagcacagtttccagagtctgcagcagggcagggctcctccctgtcctccataaaggaaaagatggctgttgtcaccctcacctcctatgtactccagggtctatggggagttctctcccctcagcgtttcaatctttctgggatcgtttcccttgcatgcctttcccttccacctccccctgcagttccgtggcagcagttaccaactttcctgaatggattcctggcccccttctccctcactatctgttggatgccctttggctcctttcctttttttggcaaaaacagtcactgtccttataaaagatttttaaattaataaaatcagtggccttcatacttggtgttctgtgtcagagagcagataggctggggagttgtgtattcccagggctgaacacgtctacttcattcgttatcagtgcagaccgaaactggtgccttctatttcccctcccaagagggagccaccctagccatgtgggctctgggcaggtgccctgGGTGTCCCAATTCCTAAACATTGTGGGAGAGAGTGTGGGTTGGCTTTTTTCTCCTGGGAGCCAGGTAGGCTGAAGGCTTCCAGATGTCAGCAGTCAGTCTAGGCAGCACCTGGTGGAAAGTCAGTGGGAGTCAAAGGTACCAGGAAGCGGGTGGATGGGATGTGGGTAAGAGGTCACCTGCCAGTCATCATAGCATGTGCTCTTAGCGTCTGGCAAGAATGAATGGGTGGGACTTTTGCTGTTTGCCAAGCTACCATAGAAAAGGAAGGAAATGGTTAAACTTGAAAGCCAAATCCTACTTCTATTGTTTTCAAGCTCATATTGTGAGCCAACCCTGAAACTAGTATCTTTGTAAGCACTTATTACATTTTTTATTTTGGAATGGTAAGAGATTTGGAGCTTAGAAAGCTAATTTGCCCTTGAAC

 

This is a a 1621 bp deletion beginning at Chromosome 19 position 5,414,502 bp and ending after 5,416,122 bp (GRCm39/mm39).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
59518 GGG CTG AAC ACG TCT ACT TCA WT Probe A
59519 Fluorophore-1 AGC CAC CCT AGC CAT GTG G Quencher-1 WT Probe
59520 CCA CAC TCT CTC CCA CAA TG Common A
59521 CGC TCT GTT GCC TTA GGT G Mutant Forward A
59522 Fluorophore-2 ATT TCC GAA TAG GTG TCC CAA TTC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
59518 0.40 uM
59520 0.40 uM
59521 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.