Protocol 31905: Probe Assay - Kcna6<em1(IMPC)J> Alternate 1
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=  127 bp

Wild Type = 142 bp

>chr6:126739793-126739934 142bp CCGAGGTGACATGAGATCG CACCAACCTCTCACTACTACAGCA

 

Sequence

Wt Sequence (deleted region in lower case):

 

CCGCCAGGTCAGAGCACGGGCCCCACCCTAAAGGAGGGCACAGCCGAAGCTGTGAAGCGGGTGCCGCTCTCTGGAGCTCGTGTCGTGGGCGCCGTCCTAGTGGCGGGGAGCGCACCGCCGAGGTGACATGAGATCGGAGAAATCCCTGAcgctggcggcgccgggggaggtccgtgggccggagggggagcaacaggatgcgggtgagttccaggaggccgagggcggcggcggctgctgtagtagtgagaggttggtgatcaacatctccgggctgcgcttcgagacgcagctgcgcaccctgtcgctgttccctgacacgctgctaggagaccctggccgcagagtccgtttctttgaccccttgaggaacgagtacttctttgaccgcaaccgacccagcttcgacgctatcctttattactatcaatctggaggtcgcctgcgcaggcctgttaatgtgcccctggacatttttatggaagagatccgcttctatcagttgggagaggaagccctggcggccttccgggaggatgagggttgcctgcccgaaggtggcgaggatgagaaaccactcccctcccagcctttccagcgacaggtctggcttctctttgagtacccggagagttccgggcccgcccgaggcatcgccatcgtctcagtattggtcatcctcatctccatcgtcatcttttgcctggagaccttgcctcaattccgtgcagatgggcgcggtggaagcaacgagggtagtgggacccgcttgtctccggcctccaggagccacgaggaagaagatgaggatgaagattcctatgcatttcctggtagcattccctctggggggttggggactggaggaacatcttcacttagtactctcgggggttctttcttcacagaccccttcttcttggtggaaactctgtgtatcgtctggttcacgtttgagctcctggtgcgcttctctgcctgtcccagcaaggcggccttctttcgcaatatcatgaacatcattgacttggtggccattttcccctactttatcaccttgggcaccgagctagtgcaacgtcacgagcagcagtctgtgagtggtggcagtggtcagaatgggcagcaggccatgtccctagccatcctcagggtgatccgcctggtccgggtgttccgaatcttcaagctctcccgccattccaaggggctgcagatcctgggtaagaccttacaggcgtccatgcgggagctcgggctgctcatcttcttcctcttcatcggagtcatcctcttttccagcgctgtctacttcgcagaggctgatgatgttgactcgctcttccctagcatcccagatgccttctggtgggctgtggttacaatgaccacggtaggttatggggacatgtaccccatgacggtagggggcaagattgtgggctcactgtgcgccattgctggggtcctcaccattgcattaccggtaccggtcattgtctccaatttcaactacttctaccaccgagagacggagcaggaggagcaaggccagtatacccacgtcacttgtgggcagcccactccggacttgaaggcaacggacaatgggcttggcaaacctgactttgccgaggcttcacgggaacgccggcccagctaccttccgactccacatcgagcttatgcggagaaaaggatgctcaccgaggttTGATGGATGCGGGCAGGCCTGCAGGAAAACAGGAGCTCTGAGCAAGTCATCTCTCAGGCTTCCTTCTCATGCTCACTACTTCCGCCTTAGCTCCAGAGGACCTCGAACCCCCCTCCCCCCTAACACAGTACATGGCATCTTGGACCAAATATCTGGACTGTAGACTGTTGCTCGATCCTCGCAGCATTCGAGGTTTCTCCATCTTAG

internal deletion beginning at Chromosome 6 negative strand position 126,739,902 bp and ending after 126,738,338 bp (GRCm38/mm10). This mutation deletes 1565 bp of ENSMUSE00000690877 (exon 1)

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
37246 CCG AGG TGA CAT GAG ATC G Common A
37248 CAC CAA CCT CTC ACT ACT ACA GCA Wild type Reverse A
37249 Fluorophore-1 TGC AGG AAA ACA GGA GCT CT Quencher-1 MUT Probe
37250 Fluorophore-2 AAC AGG ATG CGG GTG AGT T Quencher-2 WT Probe
37946 TGG AGC TAA GGC GGA AGT AG Mutant Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
37246 0.40 uM
37248 0.40 uM
37946 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.