Protocol 18267: End Point Analysis Assay - H2-T18<a/b>-JR457-EP-alternate2
Version 1.0

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

H2-T18<b> Variant = (Jr 457) = "hom"

H2-T18<b>=  (Jrs 666, 664, 438) = "wt"

Please run JR 683 as well at validation

664 and 457 controls must be run 1-18-16 ESP

 

Sequence

(T/C)TG(GT/AC)GTTCTGATGAGTTATTGATCTAG(C/T)TATGATAATCAGTGATTG(ATTG/----)CTATT(A/G)T(A/C)CACAGTCACAAGCTCAAGATTATGAAT(T/C)TCCTA(C/T)G(C/G)GTTGTCTCTAAGTATA(TA/--)GACTTAAAAGGGCTTCTCATTGAGCT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
25550 Fluorophore-1 AAT CAG TGA TTG ATT GCT ATT ATA AC Quencher-1 WT Probe A
26436 Fluorophore-2 TCA GTG ATT GCT ATT GTC ACA GTC Quencher-2 MUT Probe A
26747 TGA CGT TCT GAT GAG TTA TTG ATC T Forward A
26748 AGC TCA ATG AGA AGC CCT TT Reverse A

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
25550 0.40 uM
26436 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.