Stock No: 005783
Protocol 29075: Standard PCR Assay - Cacna1c<tm1Dgen>
Version 2.3

Notes

Optimized using Platinum High Fidelity Taq (Invitrogen) with a corresponding buffer.

 

 Use Kapa2G Fast HS Ready Mix (2X) diluted to 1X  and add Kapa2G Robust HS 5U/ul Taq. 

 Use 2ul Taq per 100ul amplification mix of  Kapa2G + primers.   S Cook   4-23-21

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 = 650 bp
Heterozygote = 400 bp and 650 bp
Wild type = 400 bp
Separated by gel electrophoresis on a 1.5% agarose gel.

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
moIMR0008 GAC GAG TTC TTC TGA GGG GAT CGA TC A
moIMR0813 CAC GAC TGG CCT CTA CTG CTC TTG AC A
moIMR0814 TCT CTC CCA CCT CGC ACG CCG AAT C A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
moIMR0008 0.50 uM
moIMR0813 0.50 uM
moIMR0814 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.