Protocol 29841 - Gria3<tm2Rlh>

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 = 190 bp
Heterozygote = 190 bp and 295 bp
Wild type = 295 bp

X-linked

Sequence

Wt Sequence: CATTTATGAGCCTGGGGATCTCCATCATGATAAAGAAGCCTCAGAAATCAAAGCCAGGCGTATTTTCATTCCTgGATCCTTTAGCTTATGAAATCTGGATGTGCATTGTCTTCG

Mutant Sequence: agactgccttgggaaaagcgcctcccctacccggtagaattcgatatcaagcttatcgataccgtcgacctcgAGATCCTTTAGCTTATGAAATCTGGATGTGCATTGTCTTCG

JAX Protocol

Protocol Primers

Primer	Sequence 5' → 3'	Primer Type	Reaction
10259	AGG AAA TGC TGT CTG TTA AAG TGT TCT GTG	Wild type Reverse	A
10260	TCC AAG TGC CAC TCA TAA GGG CTA AAT C	Common	A
oIMR9462	ATG CTC CAG ACT GCC TTG GGA AAA G	Mutant Reverse	A

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.30 X
MgCl2	2.60 mM
dNTP KAPA	0.26 mM
10259	0.50 uM
10260	0.50 uM
oIMR9462	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.