Protocol 22158: QPCR Assay - Tg(SERPINA1*E342K)#Slcw
Version 3.0

Notes

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

Sequence

 

GACAAGAAAAGGGACTGAAGCTGCTGGGGCCATGTTTTT
AGAGGCCATACCCATGTCTATCCCCCCCGAGGTCAAGTTC
AACAAACCCTTTGTCTTCTTAATGATTGAACAAAATACCAA
GTCTCCCCTCTTCATGGGAAAAGTGGTGAATCCCACCC

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
12404 AGA AAA GGG ACT GAA GCT GC Transgene Forward A
12405 ACC ACT TTT CCC ATG AAG AGG Transgene Reverse A
12406 Fluorophore-1 CCC CCC GAG GTC AAG TTC AAC AA Quencher-1 Tg Probe
oIMR1544 CAC GTG GGC TCC AGC ATT Internal Positive Control Forward A
oIMR3580 TCA CCA GTC ATT TCT GCC TTT G Internal Positive Control Reverse A
TmoIMR0105 Fluorophore-2 CCA ATG GTC GGG CAC TGC TCA A Quencher-2 IC Probe

Reaction A

Component Final Concentration
ddH2O
Kapa Probe Fast QPCR 1.00 X
12404 0.40 uM
12405 0.40 uM
oIMR1544 0.40 uM
oIMR3580 0.40 uM
Tg Probe 0.15 uM
IC Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 -- repeat steps 2-3 for 40 cycles
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

Stock Number Strain Name
035411 B6.Cg-Tg(SERPINA1*E342K)Z11.03Slcw/ChmuJ
037669 C57BL/6J-Tg(SERPINA1)1Mlb/J
037670 C57BL/6J-Tg(SERPINA1*E366K)1Mlb/J
028842 NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(SERPINA1*E342K)#Slcw/SzJ
4 strains use this protocol