Stock No: 027379
Protocol 23404: Standard PCR Assay - Tg(HTT*97Q)IXwy repeat assay
Version 1.2

Notes

AM Bufer:  670 mM TrisHCl pH 8.8, 166 mM (NH4)2SO4, 20 mM MgCl2, 1.7 mg/ml BSA, 10 mM 2-mercaptoethanol (Add 2-mercaptoethanol to 10 mM just prior to use. Do not store with 2-mercaptoethanol in buffer.)

PCR products are sized on an ABI3730.
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

FVB/NJ  = no band
If using human DNA as a control = 363 bp and 378 bp

JR8197 repeat = ~651 bp

TGS has never validated this assay

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR8074 AGG TCG GTG CAG AGG CTC CTC Reverse A
TmoIMR1594 Fluorophore CCG CTC AGG TTC TGC TTT TA Forward A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
oIMR8074 0.50 uM
TmoIMR1594 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 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

Stock Number Strain Name
027379 FVB/N-Tg(HTT*)NXwy/J
008197 FVB/N-Tg(HTT*97Q)IXwy/J
027433 FVB/N-Tg(HTT*97Q)LXwy/ChdiJ
3 strains use this protocol