Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:22051449+22051538 90bp TCTGAAAGGTTTTGGTCCATGT CTAACTTTCTTTGGAACTTTCCCC
Mut = 119 bp
Wt = 90 bp
Fam = Mut
Hex = Wt
Wt Sequence (deletions in lower case):
CTTTCTGCCAGCATATTCTTTTTTTATAAGAAGTCTTACAATTTTTTAAAAGACTAAGGTAATTTATACATAGTCTAATTTTGATTTTTCAACATAATTTTATAGATATTCAATATCTGAATTTTTCTTAGATTAAAATGCTAAATATGAAATGTTAAGCTTTTGAGTCAGAGTTGAATTTTAGTTATTCTGAGTATAAGCATTTTGCTACGAACAATGTTACTTGTGTGTTAGAGTTTAAATCATCACACTctttactggaaggagctaaatgttgtttaatgtttttaggtggtttgaactcctgggatctgttcatttgcctgccttctagggaagctgatgggaaaccctgcatacccagggagcacatgtgtcgactaagcctacatcaaagggtaaatactgccaaagcataaacacattttatcagtcctatggattaaatctgaaaggttttggtccatgtattgttgcttttaatttttgaagaattggaaccactgtatctgTGGGGAAAGTTCCAAAGAAAGTTAGTTAGCTTTCTGGTACTATAACAAAATACCTGAGATAAACCGGCATATAAAGAAGGAAGATTGACCTTTGACCCACAGTTTCAGAGTTTCAGGCCATGGTCATTTGGCCTTGCTGACTTTGTCCATGTGTTGAGGCTGTACAACATGAATGGAGTCTG
This mutation is a 260 bp deletion beginning at Chromosome 6 position 22,051,254 bp and ending after 22,051,513 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
50917 | CTA ACT TTC TTT GGA ACT TTC CCC | Common | A | |||
50918 | TCT GAA AGG TTT TGG TCC ATG T | Wild type Forward | A | |||
50919 | GCT TTT GAG TCA GAG TTG AAT TTT AG | Mutant Forward | A | |||
50920 | Fluorophore-1 | TTT GAA GAA TTG GAA CCA CTG TAT C | Quencher-1 | WT Probe | ||
50921 | Fluorophore-2 | TGC TAC GAA CAA TGT TAC TTG TGT G | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
50917 | 0.50 uM |
50918 | 0.50 uM |
50919 | 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 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |