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Mutant = 181 bp
Heterozygote = 181 bp and 249 bp
Wild type = 249 bp
>chr14:111885375-111885623 249bp CCTGGAAATGCCTCAAAATG CATGGCATGTGGTCTTCTGT
This assay is capable of distinguishing hemi from hom. A transgene insertion site is known to be on mouse Chr 14. 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 3.
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 |
---|---|---|---|---|---|---|
60927 | CCT GGA AAT GCC TCA AAA TG | Wild type Forward | A | mChr14 | ||
60928 | CAT GGC ATG TGG TCT TCT GT | Common | A, B | mChr14 | ||
60929 | TTC CGA GAG ACT CAC CAA AG | Transgene Forward | B | vector |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
60927 | 0.50 uM |
60928 | 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 |
60928 | 0.50 uM |
60929 | 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 |