Protocol 24729: Standard PCR Assay - Tg(SNCA)129Mjff
Version 2.2

Notes

This assay will NOT distinguish hemizygous from homozygous transgenic animals.

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

Transgene = 215 bp

Internal positive control = 324 bp

 

IPC                 Tm = 82      Area = 33.5

Transgene    Tm = 79.5   Area = 50   (WT*  Tm = 83   Area = 28.5

*MCA WT with Tg assay @TD50EG but expect minimal -to-no WT @TD60EG  (TD60EG  MCA plate was not exported).  SC 6-28-12

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
14909 AGA GGG AAG GCC TGA TGT GT A
14910 CAA GAT AGA CCA TAT CCT GCA CC A
oIMR7338 CTA GGC CAC AGA ATT GAA AGA TCT A
oIMR7339 GTA GGT GGA AAT TCT AGC ATC ATC C A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
14909 0.50 uM
14910 0.50 uM
oIMR7338 0.50 uM
oIMR7339 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
023837 B6.Cg-Tg(SNCA)OVX37Rwm Sncatm1Rosl/J
018442 C57BL/6N-Tg(SNCA)129Mjff/J
010710 FVB;129S6-Sncatm1Nbm Tg(SNCA)1Nbm/J
010799 FVB;129S6-Sncatm1Nbm Tg(SNCA*A53T)1Nbm Tg(SNCA*A53T)2Nbm/J
027344 STOCK Gbatm1Rlp Sncatm1Nbm Tg(SNCA)1Nbm/Mmjax
027227 STOCK Gbatm1Rlp Sncatm1Nbm Tg(SNCA*A53T)1Nbm Tg(SNCA*A53T)2Nbm/Mmjax
6 strains use this protocol