Protocol 28992: Standard PCR Assay - Ttpa<tm1Far>

B6.129S4-Ttpa^tm1Far/J

Stock No: 003823

Version 3.2

Notes

10X AM Buffer: 670mM Tris.HCL pH8.8, 166mM NH4SO4, 20mM MgCl2, 1.7mg/ml BSA. Dissolve in 9 ml TE, pH to 8.8, then bring final volume to 10 ml with TE (NOT water!) Filter sterilize. Add 2-mercaptoethanol to 10 mM prior to use.

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 = 266 bp
Heterozygote = 138 bp and 266 bp
Wild type = 138 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
oIMR1379		TGA GTG TGC GTG GGG CGG CGT CC			A
oIMR1380		CTG TTT CCC AAC CAA TGG CCC C			A
oIMR1381		CAT TCA GGC TGC GCA ACT GTT GGG			A

Reaction A

Component	Final Concentration
ddH2O
Kapa 2G HS buffer	1.30 X
MgCl2	2.60 mM
dNTP KAPA	0.26 mM
oIMR1379	0.50 uM
oIMR1380	0.50 uM
oIMR1381	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.