Protocol 36397: Probe Assay - Fam43a<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

>chr16:30599636+30599744 109bp AAAGGGATCCTCTCCTGTGC TTGCTCGCTTCACATCATTG

Mut = 107 bp

Wt = 109 bp

Fam = Mut

Hex = Wt

Sequence

Wt Sequence (deletions in lower case):

CTCTCGACCAGTGCCTGCAACCTGGTCCCTTTAAGAGACCAGAACTGCGTCCGGCGTCGCAGCCCTAAAGGGATCCTCTCCTGTGCTCCTCCCCTGTCcgcgcggtgaatggttcgtgccggcctatattaacccgagatcttcctcccggacggcaatgatgtgaagcgagcaagtcggctaccgcgcgcacgaccacagagggcgcgctgcagatcgccgagaaccccgaagcggagccggacacccccggagggaagcaatgaggtagcggcagtccccggagcggacgatgtgcgcgtcctgccctccgaaccccgcgctaccttggccgagacggccgagcgcgagtctccggcgtcccctcggagcgaggatgcagtgagctgcgggaccgctgtgcttggcccgccgcggccagccagacctctcggaccacacttggggcaagctgcttattcagggaccagtccagccaaggggctcgccctcttctgcacccactgggggtggggagcaccggcccctgggcgcactgctgtagtttccgcggctcctggccctggcccaagacttgtgtgtgtgtcacgttcgctgaagaagagttccagcttatttctctcaccatcccggttgctttggccgagccgcccacctttatttatttgtacgcactcatttggcctttttctaaaatattttccgctccgctctcttattttgttgcttgtttgagacttttgtcctggtcctcctcatctaggcttctctctctctctctctctctctctctctctctctctctctttcctgaatatttttttctaatatcctaccccagtccctgcggggttcccaagcactatctggcctctcctgcctccatctccctgccccggaggtcctcccagcctgagttttatttttcgagcttgcccgggccaagcgtagcctgggttggtggccctgcggggctctcaccccgcgctcggccaccgccgccccagccactcccgggcgtcctccggagatgctgccgtggaagaagcacaagttcgagctgctggccgaggcgccgccgcggcaggcgtccaagcctaagggctacgctgtgagcctgcactactccgcgctcagctccttggcgagagcctgcccggaaggcgcgctcagccgggtgggcagcatgttccgctccaagcgcaagaagctgcacatcaccagcgaggaccccacttacactgtgctctacctggggaatgccaccaccatccaggcgcgcggggacggttgcactgacctagcggtgggcaagatctggagcaagagtgaggccggccgtcaagggactaagatgaagctgaccgtgagtgcgcaaggcatccgcatggtgcatgccgaggagcgagcgctgcgacgtcccggccacctctacctgctgcaccgcgtcacctactgtgtggcagacgcgcggctacccaaagtcttcgcctgggtataccggcatgagctgaagcacaaggcggtaatgctacgctgccacgcggtgctggtgtccaagccggagaaggctcaggccatggctctgctgctttaccagacatcagccaacgctctggcagagtttaaacgtctcaagcggcgggatgatgcgcgtcaccagcagcaggagttggtgggcgcacacaccatcccactagtgccgctgcgcaagctgctcttgcacggaccttgctgctacaagccgccagtggagcgcagccgcagcgctcccaagctgggctccatcaccgaggacctgcttggggaacagcaagaggaggagctgcaggaggaagaagaggagcatctggaggactgcctggaggaggaggaagaagaggacggtgtgggggatggggatccagcagaggaagaggctgaggcgcagagggcgctggtggtggccatgcagttggaatgtgaggacttgctggacccgctggaaaatggccacgaggaggcgttgggggacggcggggtctcgctgggccccagctcggggacatcacttcctctgtcagtctgtgcctcagacatgaaggcccaactgtcacagcttattagtgacctgggcgaccttagctttggcaatgacgtgagcaccctggagaccgatttaagggtgacacgcctgctgtcgggcgagagcacaggcagcgagagctccatcgagggcggaggcctggatgccacccctgtcagccctggcaacccatcgggccctgctgacagcaccagcctagatgagccctactcgggctaagctgccagaagacttctctgtgcgcccctcccaacatacacatagtggctccccaacagtagttctattccccctaacctccaggaaagggaaaatgatgcacttgaagcccagacccgactttggagttttctggggaagctattccctcttgctagttgccccagcgctttggctcagaggtggctcaggttgtgaacagggggatggggggcagggagggcgagaagcactacaaaggtgggtgagggagagctggctgaagttggaaaaggccaagggcgggctggaggtgcccagcagccggtttcctgtttgtgaggcagctggggctagggaaggaggggttaacttcctctccagaccccctgactggaaggcctctttctgtcatcaatatctcattaacaaagtcactttcaaaacgttggaggcttcaatgccactttacctgcccccagttctttcggataggggagcttgggctgtttacctcagactcaaactcttgaaagtttgccaaatttctcaaataactcttcttgggggggggggcagtgtatgaaagtcgtgttttgtttacagttaaagaaacctaatatctaaaaaacaaaaaaggaattttcctttagaaatacacaccttcctctggccccaaagctcctactccatgatgctatgtaaaatatttttgcactgttgtgaagtatttttgactttttgttttttgtacataactgtgttctcggagctgaatgtttatatctcttttgctgtgcaaaagatacctgtaaaatgttgtacggttgtatatatggaaatgtgtataaaacattttgttatttttttgagcagccttgtttcagttctgttggcatgtcagcagcggggtgagaataaagaggaaaagctgtcagaacaggcgagagcctctggaaatgctaccccagatgattggcggggaggaggtgggggcggggttgtgcctccagttttagtgtggtcctgctgtggctggaagagagccggaggtgagaccttgccacccatagcgtgcttttggatcagaccaaCTCGGGGTGTTTTATCTGTGGCCAGCCTCTGTATATTTCATATTCCCATTGTGTGTGTGTGTGCAGTTTTGCTTGCTTGTTGCTTTTGAGACAAGAGACGCACTGTGCGGCTCTAACTGGCCAGGAACTTCCTAATACAGACCAGGCTGGCTTCAAATTGAGAGATCTGCTTGCCTCTGTTTCCCAAATGTTGGGGG

This mutation is a 3280 bp deletion beginning at Chromosome 16 position 30,599,668 bp and ending after 30,602,947 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
46436 TTG CTC GCT TCA CAT CAT TG Wild type Reverse A
48645 AAA GGG ATC CTC TCC TGT GC Common A
48646 CAA GCA AAA CTG CAC ACA CA Mutant Reverse A
48647 Fluorophore-1 CGC GGT GAA TGG TTC G Quencher-1 WT Probe
48648 Fluorophore-2 CTG TCC TCG GGG TGT TTT ATC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
46436 0.40 uM
48645 0.40 uM
48646 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.