Stock No: 005247
Protocol 26656: Standard PCR Assay - Nrp1<tm2Ddg>alternate1
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

The mutations in Stock# 005247 and 005245 result in similar sequence upstream of exon 2.  Stock# 005247 has both a FRT and a loxP site upstream of exon 2, while Stock# 005245 has just a loxP site upstream of exon 2, with exon 2 deleted.  The primers we use for genotyping both strains flank the FRT/loxP or loxp sequences, but will produce slightly different band sizes from the two mutant alleles.

Mutant Stock# 005247= 398 bp

Mutant Stock# 005245= ~348 bp

Wild type = 183 bp

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
19849 AAG GAG TGG CAC AGC ATC TT Forward A
19850 TCA CAC CCA AAC TTC CTT CC Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
19849 0.50 uM
19850 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

This is the only strain that uses this protocol.