Mutant = A/A
Heterozygote = G/A
Wild type =G/G
TCGAATGACTCCTCCACTCCCTGCCAGGGTGGACTTCTCCCTGGCCGGGGCGCTCAATGCTGGCTTCAAGGAGACACGGGCGAGCGAGCGTGCAGAGATGATGGA
GCTCAATGACCg/aCTTTGCTAGCTACATCGAGAAGGTCCGCTTCCTGGAACAGCAAAACAAGGCGCTGGCAGCTGAACTGAACCAGCTTCGAGCCAAGGAGCCCACC
AAACTGGCTGATGTCTACCAGGCGGAG
Information from the Donating Investigator. Please note, this protocol has not be validated by The Jackson Laboratory.
Primers detect the LoxP site in intron 3.
Forward ACA TTG AAG TCC AGA GAA GTT
Reverse TAT ATG AGG CTC TCC TGT AGA CC
191 bp = wild-type allele
309 bp = mutant allele
4.0 μl 5 x Promega GoTaq Buffer
1.6 μl 2.5 mM dNTPs
1.2 μl 25 mM MgCl2
0.4 μl 100ng/μl forward primer (pr45)
0.4 μl 100ng/μl reverse primer (pr104)
11.8 μl H2O
0.1 μl Taq (Promega)
0.5 μl Template DNA (~50ng)
1) 95oC 3 min
2) 95oC 40 s
3) 54oC 30 s
4) 72oC 1 min
5) goto step 2; 31x
6) 72oC 5 min
7) 4 oC forever
8) end
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
20392 | TCG AAT GAC TCC TCC ACT CC | Forward | A | |||
20393 | CTC CGC CTG GTA GAC ATC AG | Reverse | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTPS-kapa | 0.26 mM |
20392 | 0.50 uM |
20393 | 0.50 uM |
Glycerol | 6.50 % |
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 |