Protocol 19550: Probe Assay - Lama2<tm1Eeng> 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 transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.

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

Muant=  182 bp

Wild Type = 166 bp

Sequence

Wt Sequence: 

 

aaggggtggggactgccCTTTCTCACCCACCCTTGGGCGGTGGCAGGGCAGAGCTGGGGAGCACTGTCTCCTCCTCTTTCCCCTGCAGCTGCTCATTTCAGCTGGGTGGACC

CGAGGCTGCAACTCCGTGGGCTCCGGGAGGAGTGGATCTGCTCCGGCCAGGATGCCTGCGGCCACCGCCGGGATCctCTTGCTCCTGCTCTTGGGGACGCTCGAAGG

CTCCCAGACTCAGCGGCGACAGTCCCAAGCGCATCAACAGAGAGgtacagtccagggcgccctttccctggagcgctgctcctaacaggcttcagctagagagaaagaattgctggttggggaccactttctcg

ctttactttagggtgtgctcttccaggcgcaagggactgaactcctgcaaattaagagtaagacccccccgagagagctgt

 

Mutant Sequence:  CCTCTTTCCCCTGCAGCTGCTCATTTCAGCTGGGTGGACCCGAGGCTGCAACTCCGTGGGCTCCGGGAGGAGTGGATCTGCTCCGGCCAGGATGCCTGCGGCCACC

GCCGGGATCCacagcaaggagctgctcaagcgcgatcccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgca

ccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctttgcct

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
11358 GTT GAT GCG CTT GGG AC Wild type Reverse A
28836 CTC ATT TCA GCT GGG TGG AC Common A
28837 AGG CGA TTA AGT TGG GTA ACG Mutant Reverse A
28838 Fluorophore-1 CTC TTG GGG ACG CTC GAA Quencher-1 WT Probe
28839 Fluorophore-2 CGC GAT CCC GTC GTT TTA C Quencher-2 MUT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
11358 0.40 uM
28836 0.40 uM
28837 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles (Touchdown)
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

This is the only strain that uses this protocol.