Protocol 360 - Tg(Trp53A135V)L3Ber

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

Transgene = three digested fragments (does not have 247 bp band)
Internal positive control = two digested fragments (includes 247 bp band)

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
oIMR1237		GGA GGA GCC AGG CCT AAG AGC AA		Common
oIMR1238		ACA CCT GAT CGT TAC TCG GCT TGT C		Common

Reaction A

Component	Final Concentration
ddH2O
AB PCR BufferII	1.00 X
MgCl2	2.00 mM
dNTP	0.20 mM
oIMR1237	0.80 uM
oIMR1238	0.80 uM
DNA Loading Dye	0.69 mM
Taq DNA Polymerase	0.01 U/ul
DNA

Cycling

Step	Temp °C	Time	Note
1	94.0	--
2	94.0	--
3	64.0	--	*-0.5 C per cycle
4	72.0	--	repeat steps 2-4 for 12 cycles
5	94.0	--
6	58.0	--
7	72.0	--	repeat steps 5-7 for 25 cycles
8	72.0	--
9	10.0	--	hold

JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Reaction B

Component	Final Concentration
Hph I Buffer	X
ddH2O
BSA	mg/ml
Hph I

Strains Using This Protocol

This is the only strain that uses this protocol.