Protocol 35156: Probe Assay - Zbtb41<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

>chr1:139429332+139429494 163bp AGGCAAGTATCCTGCTCCTG AAAAGGACAATCACAGTGCATC

 

Mut= 168 bp

Wt= 163 bp

Fam=Mut

Hex=Wt

Sequence

Wt Sequence (deletion in lower case and insertion with carrots ^g^ (TTTTTTAG insertion):

 TTAGAAATACCTGAATGAGTACTTTTTTTTTTTTTTTTAAACAACCTAAGCATTTGAGGGAAAGTATACTTTAAGCATTAAGCTCAAAGTGCTGGGTCTTTGAGGTTCATTTAGTTAGAAATTTAGGTAAGTAGTTTTCTCTTTAGTT^ccctatgtgaatttgggtatatatagtaaggttattaaatggtgccttttcattgcaatgcgaagtttccgtcaaagcagagcagatacgttgaactcaattttcttctctattgtagggaaatatgagagccacacccgtgtgcacacaggtgagaagccttttgagtgtgatatttgtcaccagcgttattcgacaaagtctaacttaactgttcacagaaagaagcacagtaacgaagtagagtttcataggaaggagcacaagtgcccttactgtaacaagctgcacgcaagcaagaagacactggccaagcacgtcaagaggcaagtatcctgctcctggcgtcacacaagtaacaaatgaccaaacaaaagcaaatggcagctcgttcatgttttttctcactgttgatgaggccctttggcagt^GCAACAAGTATATGGCTGATAATGTGAAGTTTAAGATGCACTGTGATTGTCCTTTTTTTTTTTTAAGATTTTTATTTATTATTATTCATTAGTACACTGTAGCTGTCTTCAGATGCACCAGAAGAGGGCGACAGATATCATTACGGGT

This mutation is a 431 bp deletion beginning at Chromosome 1 position 139,429,008 bp and ending after 139,429,438 bp (GRCm38/mm10) . In addition, there is an 8 bp insertion (CTAAAAAA) at the deletion site..

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
44027 AAA AGG ACA ATC ACA GTG CAT C Common A
44029 Fluorophore-1 CTC ACT GTT GAT GAG GCC CT Quencher-1 WT Probe
45397 Fluorophore-2 TGG GTC TTT GAG GTT CAT TTA GTT AG Quencher-2 MUT Probe
45398 CCT AAG CAT TTG AGG GAA AGT Mutant Forward A
45399 AGG CAA GTA TCC TGC TCC TG Wild type Forward A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
44027 0.40 uM
45398 0.40 uM
45399 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.