Genotyping Protocols Database
Stock No: 008180
Protocol 29388: Standard PCR Assay - Kras<tm4Tyj> Alt1
Version 1.2

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

Mutant (embryonic lethal) = 100 bp
Heterozygote = 100 bp and 250 bp
Wild type = 250 bp

 

After Cre-recombination a bandsize of ~300 is expected.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
22907 TGT CTT TCC CCA GCA CAG T Wild type Forward A
22908 CTG CAT AGT ACG CTA TAC CCT GT Common A
oIMR9592 GCA GGT CGA GGG ACC TAA TA Mutant Forward A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
22907 0.50 uM
22908 0.50 uM
oIMR9592 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
008179 B6.129S4-Krastm4Tyj/J
008180 129S/Sv-Krastm4Tyj/J
019104 B6N.Cg-Krastm4Tyj/CjDswJ
032429 STOCK Krastm4Tyj Trp53tm1Brn Tg(Pdx1-cre/Esr1*)#Dam/J
032435 B6.129-Krastm4Tyj Trp53tm1Brn/J
5 strains use this protocol