Protocol 34640: Probe Assay - Polr2l<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

>chr7:141473421-141473546 126bp CTGGTGTCAGGTTTCAATCG GAGGCTGGATGTGGTCTCT

Mut= 114 bp

Wt= 126 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AGACTGGCACATGAGCAGGTGTAACTTCTAGGTGGGGAAAGGGTCACCTGGCTCCTCCCTGAGCATCTAGATACATATTTCAGAAATCTCCAGGCCATTTCCCTATGGCCTGGTGTCAGGTTTCAATCGCTGATTATGTAGATGTGCTCTGGTAACCCCTATCCACACGAATACCcagaggcaagctggaggtggttccaaacctatccagactctagagaccacatccagcctcactcttctctttacagggatgccctggacgctctgggcctaaagcgctactgctgccgccgcatgctgctagcacacgtggacctgattgagaagctgctgaactatgcacccctagagaagtgaccgctggagcctgccctcctctgtctcctgctggccacgggcagcctgtcgtgctatgttcaaactgagctattggggatgttggtgtattgcctggctgtggccctgtggctaagtcctgttaccctggaaggaactgtttagtaaagatgtttgcagaaccaccaacaggtctaacttccaaggtgctccatgtccatgtctgtgctgtctctgaggctacctggcctgcctccagtctttcccctagggctggtctccttccccttcatctgactgggagtctcgtcctgtggaggagtaccctgcttccgttccccggggccttgaccactttgatcaacgaggtagccctacagtagtcttgtaccacattgctttagggcctctccgcttcctgcctgtgttcaggatgggcataatggatgtgggcaccacaccatccacaggtatggatgggcccaagcctggtgacaatattcctttggaatgctgagcgtgcttggaggtcccaatccttgtgctagggttattatgcatttgggtatttggtctgcttataagtctttgtatcactgtgtccccacagaggccagatcccctgagagtaaagttatagaatgtataagctgccatcaggtattgggaatcaaacccagatttatttacctttcattttccaagacagggtttctctgtttggccttgactatcctggaattcaactctgcagaccaggctggccttgaactaaagagatcagtctctgcctcccgagtgttgagtttaaaggacggcttgtgccacccaaccaaacccagatactgtaggagtacgcccaaggccacaggcctcacgaatttcttgggtcctaaagcccatcagctgtgtgagcctatctgctgcccatgcctgaggatcttgtttccagaatcttcccaggcccgacagctaggcagccctgccccttgacccaatccccacagcatgtagtcctaagaagcctactctaggccaaaggaggtggtgtagtgctttagctcctaggcatctgcatcaagtaccccgggttctttctagcacattgatccctttgagaattttggtgggtttttttgttttgttttttgagaccagctttggctggtttgaaactcacaacagtcagcctgcctttacctcttggattctgggattaaaggtgtgtgtggcatagcatacccaccctaccccacccctggttttttgttgttttaaagaaaaaggcgggctggtgagatggctcagtgggtaagagcacccaactgctcttccaaaggtccagagttcaaatcccagcaaccacatggtggctcacaaccatccgcaacaagatcctcttctggagtgtctgaagacagcaacagtgtacttacatatattaataaataaatctttaaaaaaaaaaaaaaaagaaaaaggctttgccttaggctagagtccagagaacccatagtggcctcctccagctggcacagaagactgtggggaacaaaatcagcttaggacctcttctcagctgcttagatgagctaaagccagagaaacaggagccagggcatccccaacaggccctggtgcctcccttgccctctgaccctagcAAGGCCTACAGACTCTTCCTTGAGCATGGACAAGTCCAGGCCACAGTGGGCTGGGGGGATGTTCCAGCTACCAGTGCTCACCCTAACTGTAATTGCCATTGGGCTTCACCAACCTTGACTCTTGTGAGTACTCCAGCTACCCTGGACCATAAGGTCCTGTCA

This mutation is an 1828 bp deletion beginning at Chromosome 7 position 141,471,653 bp and ending after 141,473,480 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44098 CTG GTG TCA GGT TTC AAT CG Common A
44099 GAG GCT GGA TGT GGT CTC T Wild type Reverse A
44100 CAC TGT GGC CTG GAC TTG T Mutant Reverse A
44101 Fluorophore-1 AGG CAA GCT GGA GGT GGT Quencher-1 WT Probe
44102 Fluorophore-2 ACG AAT ACC AAG GCC TAC AGA CT Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44098 0.40 uM
44099 0.40 uM
44100 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.