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The mutations in Stock# 005247 and 005245 result in similar sequence upstream of exon 2. Stock# 005247 has both a FRT and a loxP site upstream of exon 2, while Stock# 005245 has just a loxP site upstream of exon 2, with exon 2 deleted. The primers we use for genotyping both strains flank the FRT/loxP or loxp sequences, but will produce slightly different band sizes from the two mutant alleles.
Mutant Stock# 005247= 398 bp
Mutant Stock# 005245= ~348 bp
Wild type = 183 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
19849 | AAG GAG TGG CAC AGC ATC TT | Forward | A | |||
19850 | TCA CAC CCA AAC TTC CTT CC | Reverse | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
19849 | 0.50 uM |
19850 | 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 |