Protocol 33186: Probe Assay - Emilin2<em1(IMPC)J> Alternate1
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

>chr17:71280572-71280660 89bp CATTGGTACCCTGCTGTGTG AGCATCTGCTCAGTCGTCCA

Mut= 90 bp

Wt= 89 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case; and insertions with carrots ^g^ (CTTAGA insertion):

 

CTGACTCCTCCAGCTAGGCTCCACTTAAGGCCTTTAGAAGCTCCCCCCCCCCCATAGTGCTACCATTTAGGGACAAAGTATTTATAGCAGAGCCTATGCAGGACATTTCACATCACAACAAGGGTCAGCATGGGAGGACTGG^agactctcttaaaactagtggtcactaaagtgagaccctgttttctctgtctttaggtatcgggtaaactttagacccagatttgtcactagatacaagatagtgacacagttggaatggagatgctgtcctggctttagaggaccagactgccaagaaggtcccaaagaccacatgaagaccccccggcccccatcagctcgaccaaaaaacaacctgaagaaagccacaggtaatgaatgtctcattggtaccctgctgtgtgtggtcacatttgatgtccctggtgttgttaccaag^GACAGGGTTGATAATGGACGACTGAGCAGATGCTCATGTAGAGGGATTGTTATCTGAAGGGGCAGGAGTGGTTGGGTCGCACATATTTCCTTGCAAGTAAATTTCCTCGTTAGGGCCACTTGTAGATTAGCCAGGAGTGGTGGTACACATCTGGGAGGT

This mutation a 300 bp deletion beginning at Chromosome 17 position 71,280,606 bp and ending after 71,280,905 bp (GRCm38/mm10). In addition, there is a 6 bp insertion (TCTAAG) at the deletion site, that will not alter the results of the exon deletion. 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
39942 CAT TGG TAC CCT GCT GTG TG Wild type Forward A
39943 AGC ATC TGC TCA GTC GTC CA Common A
39944 CCT ATG CAG GAC ATT TCA CAT C Mutant Forward A
39945 Fluorophore-1 CAC ATT TGA TGT CCC TGG TG Quencher-1 WT Probe
40859 Fluorophore-2 ACT GGC TTA GAG ACA GGG TTG A Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
39942 0.40 uM
39943 0.40 uM
39944 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.