Protocol 37511: Probe Assay - Gtsf1l<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

>chr2:163086742-163086845 104bp CAGCCACACGCAGAAAATG GCAACATAGTTTCCTTAGTTAGGC

Mut = 113 bp

Wt = 104 bp

Fam = Mut

Hex = Wt

 

Sequence

Wt Sequence (deletions in lower case):

ACAAGTCTTTCTCCGACTTCTAAGTGTACCCCCAATGTCACTCCTTACAAATAAACACATGATGTAAAAAAGATGTTTTTAAAAGACCACACAACCGAcgctgatgaataaggggtttgattatgctaatgaagcccgaaactgccaagggccgggtacgtgtcataactacatttgaacagcagctttcctcaagccacttcccccacccagtggacagtctgtccctgatgtcataagctgcccattgtgaccacctgttctgtcctttgacaacacagggtatcttagcgtcaaagcatcattataacctgctaacttcacctccttggaagctactgtttcgggctcttgtacctgcctgagttaacgtctctggtggctgttttctggctttctctgcatagcgattgctgacagaacgtgagtgagaatttcactccagagacccaagagccaaagcctggcagaagacagcacaatctttcacaggaagaggcttttcctttcgaggtgtaccacgtacactttgggcagcaggacacttggcttcaagtcaactcccggatggagccagaatccatagaaatttgtccttataaccctcaccaccgaatcccgctcagcaggttccagtaccacctggcgtcatgcaggaagaagaaccccaagaaagccaaaaagatggccagctgtaaatacaacgcctgccacgtggttcccatcagaaagctggctgaacatgaagctacctgtgtcaacaggagctccgtggaggaagaggacacattaggccctctgcaagtcagcctcccacagccgcagaaccaggacacactacaggttcgttggctttccaaccctgacatttggaatgttgacggcgccaactgtcacccaatgttcgtccttaagagttttgttccccaaaaacttgtttgtgaaagtgacatccaagagtcacggggaggagaccaatgcccagaagatcctcagactaggaccaggaaggcaaacttctagcaggaggaagagggctctcaaatgcgtggatggatcgcgcatctcatctgaataaaaacctgcaaagtaacggtccgggctgtgctggttgtttttaaaggtttagaaagagcggtggaggtggggtttagaactgagctgagaatgtaaattgaaccaccttccatggggaacacttaacagtcgctatgaaaatgctcactcaacgcacacaggctttgatcaagtcattcgacatctaaaacagcatcccgcacagccagccacacgcagaaaatggggttgagtaaaaagttactcccgtgttatttatagcagcactgaggtaccCATAGGGAAGGCCTAACTAAGGAAACTATGTTGCAACTTTACAAAGGAGAGA

This mutation is a 1255 bp deletion beginning at Chromosome 2 position 163,086,776 bp and ending after 163,088,030 bp (GRCm38/mm10). There is a 3 bp insertion (TTA) 5 bp after the deletion site.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
51525 GCA ACA TAG TTT CCT TAG TTA GGC Common A
51526 CAG CCA CAC GCA GAA AAT G Wild type Forward A
51527 AGT GTA CCC CCA ATG TCA CTC Mutant Forward A
51528 Fluorophore-1 ACT CCC GTG TTA TTT ATA GCA GCA Quencher-1 WT Probe
51529 Fluorophore-2 AAA AGA CCA CAC AAC CGA CAT AGT T Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
51525 0.40 uM
51526 0.40 uM
51527 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.