Genotyping Protocols Database
Stock No: 002667
Protocol 26240: Separated MCA Assay - Nnt<C57BL/6J>
Version 4.2

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      Tm = 83  
Wild type  Tm = 82  

Mutant = 743 bp (B6 664)

Heterozygote  = ~570 bp and 743 bp

Wild type = ~570 bp (FVB)

This allele contains a stretch of 17,814 bp missing between exons 6 and 12 in B6 mice.  For this assay B6 is the mutant.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
oIMR7332 GTA GGG CCA ACT GTT TCT GCA TGA Common A, B
oIMR7333 GGG CAT AGG AAG CAA ATA CCA AGT TG Wild type Forward B
oIMR7334 GTG GAA TTC CGC TGA GAG AAC TCT T Mutant Forward A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
oIMR7332 0.50 uM
oIMR7334 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.

Reaction B

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
oIMR7332 0.50 uM
oIMR7333 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
007083 B6.Cg-Cav1tm1Mls/J
002667 C.129S2(B6)-Cd28tm1Mak/J
012371 C57BL/6-Del(12Bdkrb2-Bdkrb1)1Mki/J
000664 C57BL/6J
002404 STOCK Mostm1Ev/J
5 strains use this protocol