Stock No: 008320
Protocol 18785: Standard PCR Assay - Lepr<tm2(cre)Rck>-Alternate 3
Version 1.0

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 = ~500 bp
Heterozygote = ~500 bp and 400 bp
Wild type = 400 bp

This is additional information from the DI-TGS has not validated these primers/conditions at the Jackson Laboratory:


Wild-type forward: 5'- TGCACATTCCCAGCCCAGTGT
Mutant forward: 5' - CACGACCAAGTGACAGCAAT
Common reverser: 5' - GACAGGCTCTACTGGAATGGA
The annealing temp is 60 degree for all primers. The wildtype amplicon is about 220bp and the mutant amplicon is about 850bp. Elongation is 1min. Since the two bands are quite different in size, I recommend that the two PCR reactions are run separately."

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
26994 GCT GGA AGA TGG CGA TTA GC Mutant Forward A
27018 CCC AAT TTC AAA CCT GTT CC Wild type Forward A
27020 TCT TCT TTC CAG AGT TCA GAT GT Common A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
26994 0.50 uM
27018 0.50 uM
27020 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.