Stock No: 004938
Protocol 8955: Fluorescent PCR Assay - HDexon K2G CAG repeat
Version 5.0

Notes

Size on ABI 3730.

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

JR6471 Transgene = ~596 bp

JR4938 Transgene = ~500 bp

JR2810 Transgene = ~ 724 bp

JR6494 Transgene = ~ 574
C57BL/6J = no band
Human = ~ 266 bp and 280 bp

There are 168 bases upstream and 53 bases downstream of the repeat for a total 221 bases to subtract to get the repeat length.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR6533 GGC TGA GGA AGC TGA GGA G
TmoIMR1594 Fluorophore CCG CTC AGG TTC TGC TTT TA

Reaction A

Component Final Concentration
ddH2O 0.00
Kapa2G Buffer M (wit 1.00
MgCl2 1.20
dNTPS-kapa 0.20
TmoIMR1594 0.28
oIMR6533 0.28
Betaine 0.50
DMSO
KAPA2G Fast HotStart 0.01
DNA (crude prep dilu

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 --
4 68.0 -- repeat steps 2-4 for 10 cycles, decreasing annealing temp 1.5 degrees C for each subsequent cycle
5 94.0 --
6 50.0 -- hold
7 72.0 -- repeat steps 5-7 for 28 cycles total
8 72.0 --
9 10.0 --
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
006471 B6.Cg-Tg(HDexon1)61Gpb/J
004938 FVB-Tg(YAC128)53Hay/J
2 strains use this protocol