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Mutant = 276 bp
Heterozygote = 233 bp and 276 bp
Wild type = 233 bp
>chr3:126382928+126383160 233bp TTGTCTCCTCAATGAAGTGAAGT ACAGACAAGGCAAACCTGCT
This assay is capable of distinguishing hemi from hom. A transgene insertion site is known to be on mouse Chr 3. JR10799 was used to determine this transgene insertion site, however, JR10799 contains both Tg(SNCA*A53T)1Nbm and Tg(SNCA*A53T)2Nbm. A second insertion site is known to be in mouse Chr 14.
This assay is NOT able to be used for copy number evaluation. If this is required, it is suggested to type by qPCR.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
58235 | TTG TCT CCT CAA TGA AGT GAA GT | Wild type Forward | A | mChr3 | ||
58236 | ACA GAC AAG GCA AAC CTG CT | Common | A, B | mChr3 | ||
58237 | TCC CCT TGA TCT CCT TTT GT | Transgene Forward | B | PAC |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
58235 | 0.50 uM |
58236 | 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 |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
58236 | 0.50 uM |
58237 | 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 |