Protocol 27441: Standard PCR Assay - Ascl1<tm1.1(Cre/ERT2)Jejo>
Version 3.2

Notes

This assay does not work well without the use of a Hotstart Taq polymerase.

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 =~300 bp
Heterozygote =~300 bp and 418 bp
Wild type = 418 bp

 

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
10653 CGC CTG GCG ATC CCT GAA CAT G Mutant Reverse A
10841 TCC AAC GAC TTG AAC TCT ATG G Wild type Forward A
10842 CCA GGA CTC AAT ACG CAG GG Wild type Reverse A
10843 AAC TTT CCT CCG GGG CTC GTT TC Mutant Forward A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTP KAPA 0.26 mM
10653 0.50 uM
10841 0.50 uM
10842 0.50 uM
10843 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.