Protocol 34887: Probe Assay - Syt8<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

>chr7:142440520+142440605 86bp TCTCTGCTCAAGGCCTGATTC TCCTGCTCCTCTACCAGTCC

Mut= 95 bp

Wt= 86 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case):

 AGGGGGTGGGGGGGTGCAAGGTTCAAGCTGGGGAGCTCAAGGCGGCCCTGGCAGGCCTTCTTCTCAGAGGAAGGAGATGAGCAGAGGTGATAGGGAACGAGTGATTCAGGAAGCAGGCAGATGTTGGTCCATGGTCcattggaagctacagtagtagcagtagtagcatatatgtcacattgaaggaagattggctggtctcagggagctgatctgtgttacaatagtccagatctctggttaaagattggtctcaatcatacttgcctgatctcccaccatccaccccaggttcaaccagatgtggactgcctagaaccctgctctggaggggaccaacagtggggtcgactcctgctgtcactagagtatgattttggaagccaggaggtaaggctcactcagcaggtccagaggctctgtggttgatggagaatgtgatgaatgaagagtcaggtagaggggagcaggggaaaagaacctcttgaccccaggctgagccaacccactggccctcagattagagtgggcctgaggcaggctggaaacctgaaggctgagggcacagcagacccctacgcctgggtcagtgtgtccacccagtctgggagaagacatgagacaaaggtgcaccgagggacgctctctcccatgtttgaagagacatgctgctttctggtaagtctagtctgggtagatagagtgggctggagccagggcagcaggacccagctcattaccctgtctgcaggtcccaccagcagagctgcccaaagccactctgaaggtgcagctgtgggacttcaagcggttttcagaacacgagccactgggtgagctccaactacccctgggcaccgtggaccttcaacatgtcctggagagctggtaccagctaggccctccagggaccactgaggtgagcctggctggtaggggcccgggatctacctgtaacatggggggaggacaaggtctgaatgctgtacctgctgagttcaaacccccaagggtaaggatgaccctcccagttgcagccccgtcttgttgcccatagcctgagcagatgggggagctgtgcttctcactacgctacgtgcccagctcaggcagcctgactgtggttgtactggaggctcggggtctgaatccggggcttgcaggtgagagctatacttttgtggacagcgagctggaccctcgtgtgcatgtatgggactcccttcttgaactttctcactcgtgcagaggcctacgtgaagatccagcttatgttaaaccagagaaaatggaagaagagcaaaacatcctccaagaaaggcacgactaccccctacttcaacgaggccttcgtcttcctggttcccgttagccagctccaggtagggagctgggaggaagggcagagtggcctgggacgttgtgccatgccaaactgacaaccatcccctctacatgtctgcagagtgtggacctggtgctggctgtctgggcccgtggtctgcagctccggactgagcctgtgggcaaagtgctgctgggttcccgggcttctggccaacccctacagcactgggcagacatgctggcccatgctcggcggcccatcgcccagtggcaccacctgcggtcccccagggaagtggatcgtgttctggccctgcagcctcgccttcccctgcttaggcctcgctcctgaaagcaccgcgggtctcagttttcccagatggagagctgtggatatttgtgcaggctcttcagatctctctgtaataaacaaattctctttcacactcacatgtcctgatggagccctgtgaggaggatctgagatacaaggcaggctacactttggcaccactgtcctctcaaggttggggtagggtgctctgaacagatttgccagtcctgggcctctctctgctcaaggcctgattcctaggcctttccctgaaaaacgggcacCACCCTCTCTGCCAGCCAGGACTGGTAGAGGAGCAGGAGGCTAGAGCTGGCTCTGTGAAGGCAAAGAGGGCAATTGAGGGGGAGTTGTGGGTAGGTACAGCTTGTGGAAAAGACCTCAAGACCATGAGGGACTGTGATACAGTG

This mutation is a 1829 bp deletion beginning at Chromosome 7 position 142,438,739 bp and ending after 142,440,567 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44709 TCT CTG CTC AAG GCC TGA TTC Wild type Forward A
44710 TCC TGC TCC TCT ACC AGT CC Common A
44711 AGC AGA GGT GAT AGG GAA CG Mutant Forward A
44712 Fluorophore-1 TCC CTG AAA AAC GGG CA Quencher-1 WT Probe
44713 Fluorophore-2 CAG ATG TTG GTC CAT GGT CC Quencher-2 MUT Probe

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
44709 0.50 uM
44710 0.50 uM
44711 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
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.