Protocol 31520: Probe Assay - Ccdc191<em1(IMPC)J>
Version 2.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

>chr16:43943235+43943339 105bp AAACCAAAAGCACCATCAGC GCCAATCGGAATGTACAACTG

Mutant=  91 bp

Wild Type = 105 bp

Sequence

Wt Sequence:  GGCAAGGGGAAGCAGGAAGGTTAATTCCAGGACAGTCAGAGCTCTATGAAATGCTGTCTCCAAAAACCAAAAGCACCATCAGCCTTGGCCCTggcagcagccgtcacagctctgaccactctccccacttacacctgaaggtaatcccagttgtacattccgattggctcagcgtgccaaggctgatggtgtctgaggcactcaggcctggagaagggcagctctagctgaggtgcagcgtagctgtgccactgaacagaacaataccagtgagaccacagcaggagcttgtctcatcagtgtttgagttagaatgtcatcttttcccctaggtcactatcctgtcccctttgtgggaaaggccacctgtggagagcagtggttgtacactcagccattccccagaaagaaccaccaagggtaattgtcagggtcctctcccagatgtctctgggagcacacctggcacggagcagtgcaaggctgtagcagctgagacctctgagcagcctggtagctgtgaacgacctgggaccacaaggcaggaggcagaagctgcttgtgtgaggcatttccaccgtagccatgtgtaccagcaacagctgattgagaagcaaaagaagaaacttgagcagcagcagaaaaccattcagaagctaaaggaaaaccagcgcttggccgaggcccggtgggcagctaagcgagccccggcagtcacagaaggacagaactgccttcagtcagaccccagaggagcaagggacccgcagggcacctgccagaggcttctgaagtatgtcagcagggtcacagcactcggaatgtgtttgcagggttttcctctaagctcagtagctttgcttggcttcctgtatgctcacaaagccaggaacctcatgtatgctgactgtcccatgccaggtgtacagagataagcaaatggccgcctagacggataccattttacagtgagccctgggtggttgtgagacaaacactggaattatgtaggaaaagagaaaaatacgtgttctaagcttttgggtttctgatggtttaatgctgagaaacgagGGCAGACAGCATGGTCCCCAGGAGCAGCCCCAGGGCTGGCTCATTGACTCACTTGAATTTTCAGTTGTTCACAGTAAAAC

 

Deleted Region: ggcagcagccgtcacagctctgaccactctccccacttacacctgaaggtaatcccagttgtacattccgattggctcagcgtgccaaggctgatggtgtctgaggcactcaggcctggagaagggcagctctagctgaggtgcagcgtagctgtgccactgaacagaacaataccagtgagaccacagcaggagcttgtctcatcagtgtttgagttagaatgtcatcttttcccctaggtcactatcctgtcccctttgtgggaaaggccacctgtggagagcagtggttgtacactcagccattccccagaaagaaccaccaagggtaattgtcagggtcctctcccagatgtctctgggagcacacctggcacggagcagtgcaaggctgtagcagctgagacctctgagcagcctggtagctgtgaacgacctgggaccacaaggcaggaggcagaagctgcttgtgtgaggcatttccaccgtagccatgtgtaccagcaacagctgattgagaagcaaaagaagaaacttgagcagcagcagaaaaccattcagaagctaaaggaaaaccagcgcttggccgaggcccggtgggcagctaagcgagccccggcagtcacagaaggacagaactgccttcagtcagaccccagaggagcaagggacccgcagggcacctgccagaggcttctgaagtatgtcagcagggtcacagcactcggaatgtgtttgcagggttttcctctaagctcagtagctttgcttggcttcctgtatgctcacaaagccaggaacctcatgtatgctgactgtcccatgccaggtgtacagagataagcaaatggccgcctagacggataccattttacagtgagccctgggtggttgtgagacaaacactggaattatgtaggaaaagagaaaaatacgtgttctaagcttttgggtttctgatggtttaatgctgagaaacgag

982 bp deletion beginning at Chromosome 16 positive strand position 43,943,264 bp and ending after 43,944,245 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37043 AAA CCA AAA GCA CCA TCA GC Common A
37044 GCC AAT CGG AAT GTA CAA CTG Wild type Reverse A
37045 GAA AAT TCA AGT GAG TCA ATG AGC Mutant Reverse A
37049 Fluorophore-1 AGA CAG CAT GGT CCC CAG Quencher-1 MUT Probe
37050 Fluorophore-2 AGC TCT GAC CAC TCT CCC C Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37043 0.40 uM
37044 0.40 uM
37045 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.