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Mutant = 384 bp
Heterozygote = 384 bp and 142 bp
Wild type = 142 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
21306 | CTG GCT TCT GAG GAC CG | Wild type Forward | A | |||
24500 | CAG GAC AAC GCC CAC ACA | Wild type Reverse | A | |||
24951 | AGG GCG AGG AGC TGT TCA | Mutant Forward | A | GFP F | ||
24952 | TGA AGT CGA TGC CCT TCA G | Mutant Reverse | A | GFP R |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
21306 | 0.50 uM |
24500 | 0.50 uM |
24951 | 0.50 uM |
24952 | 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 |
---|---|
013205 | 129S-Gt(ROSA)26Sortm1(NOTCH3)Sat/Mmjax |
013207 | 129S-Gt(ROSA)26Sortm2(NOTCH3*C455R)Sat/Mmjax |
006148 | B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J |
005670 | B6.Cg-Gt(ROSA)26Sortm1(rtTA,EGFP)Nagy/J |
013206 | B6;129S-Gt(ROSA)26Sortm1(NOTCH3*R1031C)Sat/Mmjax |
018422 | B6;FVB-Tg(Aicda-cre)1Rcas/J |
036418 | B6;SJL-Gt(ROSA)26Sorem1(TAg)Sac/J |
031010 | STOCK Gt(ROSA)26Sortm1(rtTA,EGFP)Nagy TgTn(pb-tetO-Pou5f1,-Klf4,-Myc,-Sox2,-mCherry)250Nagy Tg(Pou5f1-EGFP)1Nagy/J |
031012 | STOCK Gt(ROSA)26Sortm1(rtTA,EGFP)Nagy TgTn(pb-tetO-Pou5f1,-Klf4,-Myc,-Sox2,-mCherry)250Nagy/J |
005572 | STOCK Gt(ROSA)26Sortm1(rtTA,EGFP)Nagy/J |
10 strains use this protocol |