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Melt Peaks
Wt = 87.4°C +/- 1
Mut = 79.8°C +/- 1
Mutant = 103bp
Heterozygote = 103bp and 143bp
Wild type = 143bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
oIMR7033 | TGG GAG TCC ATC CAT CCT AAG TC | Wild type | A | |||
oIMR7034 | GCT AAG AAA ATG ACA ATT GCA CAT TTG | Wild type | A | |||
oIMR7035 | GGC AGT TTT AGA CTC ATC ATG TAT CTG | Mutant | B | |||
oIMR7036 | ACT TAT GGA GAT CCC TCG AGA TAA C | Mutant | B |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
oIMR7033 | 0.50 uM |
oIMR7034 | 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 |
oIMR7035 | 0.50 uM |
oIMR7036 | 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 |
---|---|
007966 | B6.Cg-Smn1tm3(SMN2/Smn1)Mrph/J |
007249 | B6;129-Smn1tm3(SMN2/Smn1)Mrph/J |
008704 | B6;129-Smn1tm6(SMN2)Mrph/J |
007964 | FVB.Cg-Smn1tm3(SMN2/Smn1)Mrph/J |
008783 | STOCK Grm7Tg(SMN2)89Ahmb Smn1tm3(SMN2/Smn1)Mrph Tg(CAG-cre/Esr1*)5Amc Tg(SMN2*delta7)4299Ahmb/J |
007951 | STOCK Grm7Tg(SMN2)89Ahmb Smn1tm3(SMN2/Smn1)Mrph Tg(SMN2*delta7)4299Ahmb/J |
6 strains use this protocol |