Protocol 34489: Standard PCR Assay - Cacna1e<tm1.1Tsch>
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

>chr1:154701230-154701390 161bp TCTGTTTCACGCCATTTGAG TCAAAGGAACCCAGAGGAGA

 

Mutant = ~250 bp
Heterozygote = ~250 bp and 161 bp
Wild type = 161 bp

Read-through band of 1190 bp may be seen.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
43756 AAG CCT TGC TTC AGG AGT CA Mutant Forward A
43757 CAA CAG TCC CTT GGG AAA GA Mutant Reverse A
43758 TCT GTT TCA CGC CAT TTG AG Wild type Forward A
43759 TCA AAG GAA CCC AGA GGA GA Wild type Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
43756 0.50 uM
43757 0.50 uM
43758 0.50 uM
43759 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.