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This assay tests for the presence of the retinal degeneration Pde6brd1 mutation.
The WT-specific primer, oIMR2094, sits on a known SNP identified as rs47440869, located at the nucleotide indicated in red: ACC TGC ATG TGA ACC CAG TAT TCT ATC. Information can be found at http://www.informatics.jax.org/snp/rs47440869.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
oIMR2093 | AAG CTA GCT GCA GTA ACG CCA TTT | Mutant | A | |||
oIMR2094 | ACC TGC ATG TGA ACC CAG TAT TCT ATC | Wild type | A | |||
oIMR2095 | CTA CAG CCC CTC TCC AAG GTT TAT AG | Common | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
oIMR2093 | 0.50 uM |
oIMR2094 | 0.50 uM |
oIMR2095 | 0.50 uM |
Glycerol | 6.50 % |
Dye | 1.00 X |
Kapa 2G HS taq polym | 0.03 U/ul |
DNA |
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 |
Stock Number | Strain Name |
---|---|
002810 | B6CBA-Tg(HDexon1)62Gpb/1J |
001924 | B6EiC3Sn a/A-Ts(1716)65Dn/J |
004850 | B6EiC3Sn-Rb(12.Ts171665Dn)2Cje/CjeDnJ |
004861 | B6EiC3Sn-Ts(16C-tel)1Cje/DnJ |
006554 | B6SJL-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax |
004828 | FVB.129P2-Pde6b+ Tyrc-ch/AntJ |
6 strains use this protocol |