Genotyping Protocols Database
Stock No: 003008
Protocol 29316: Standard PCR Assay - Tnf<tm1Gkl>
Version 4.3

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

MCA Preferred

65-60

WT TM = 87

MUT TM = 89

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR4182 TAG CCA GGA GGG AGA ACA GA Common A
oIMR4183 AGT GCC TCT TCT GCC AGT TC Wild type A
oIMR7297 CGT TGG CTA CCC GTG ATA TT Mutant A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
oIMR4182 0.50 uM
oIMR4183 0.50 uM
oIMR7297 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
005540 B6.129S-Tnftm1Gkl/J
003008 B6;129S-Tnftm1Gkl/J
007082 CByJ.129S(B6)-Tnftm1Gkl/J
3 strains use this protocol