Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr9:106451272+106451365 94bp GGCTGTGGGCATGAATGA AACGGCAATGAATGGGAAG
Mut= 92 bp
Wt= 94 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
CTGATAGACTCCAGAGCTTCATTCTTTTCAGGACACTGTTAGAACGAACACAAACATAAAAAGAATTGTCTCATTCTCGCCATAGATAAAAAACACTGGGCTGTGGGCATGAATGAATTTCAGTCTACTACTGGGTGTTACGTGAacctgggcacagagtggaggaagggacccttcccattcattgccgttgtctctcctacgcccgactacctgcaggtcttgggcgatccaaggaagcagcagcccctgcccctattggggaaccggcccctggcagcttcctagctgctgtggtggataccttggagctgggacccccagttgtgatcagtccatcattaagtggcatgtattcactgcccttcctcgtggcccccgggtcccaactccggggctttgtaccagtggctcctatctgcacagacaaaatcaatgctgtggactacgccagtgtgaaggtaccgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgtgactgagatggtatctcaggagcaagtcggggtccatccctgggccttcttcatctgggagctgggtaaccaatggggtggtttctggggagaccccttttggttgacttagctgattgtttcgccaatccctgtataagactgtgtgctggggaaaccacaaaccaaggtctccatccactgcatcagcaccgcctctcctcctcaggagggcaggaagagactgaagaggtggtgaggcctgaagctaacctttagaagctgggctggtacgaccagcccccgattctgttctcctttccttcccctcagactccagctctcattgtatatggagaccaggaccccatgggctcctccagctttcaacacctgaagcagctgcccaaccacagagtgctggtcatggaaggggcagggcatccctgttacttggacaagcctgatgagtggcataaggggctgctggatttcctgcaagggctagcctgagactcagtcctgatgataggaggttacagactgcctgctctaggatctcttgctgtcttctcctggtctctggcttctgcctcacctgcaacaggtctgtctatacatctgtctgggttgctgtcatttctgtttttctcttgcagtgacagatcagatcaaggcggtaaatcaagtggaaaaagcgtcgggttcaaggggagaactctgattgtgggtaatccataagcttcctctacaggctccccgcttggttctcacttctgccttacttcactttctgatccatctctctcttcagccgctccacaggacagccattccttctgccgcccaccccccttagaccacagacagcatatgggtattgcttgtttagatccatcccacacccagacagaacctttcccttgggagcagcacaggggtcccacattcttgtgtagtttgcacaggtacatgtgcagtttcctgaaggatggagggaatctttgtaaacagagatccacttgccaggataaaaatgtggccaccgctagatttctcttacctacccgttgcaactggtcccagaccagaatggcctcctcagaaggggaggtgctcttcaaatctcccttaggggctgcatacctccccaggtaatcaagcacggatcttctcagccaagcccatttcctgccctgaggtttgggagtgggataaggagaggagaccgacttcggaggtccctgaaataaagtgatacaattagacctcacatgcttttgctgtctccggagcgccgcccctcccccttccccttgggactgccactgtgggctcctcccccaccctggcgggaccaaatggtccccctgccctttgtccactaggccagatggagggaaagggagattggattcccctccccacccacatacatccaggtgccaagagactcagctgaggggggaggtgtaggtcagtgggacctatatgagccgattactgaaagggggagaatatctgctctgagatgggggaagggcaggctcaattcccccccccccccacacaccCACCCACACACATATACACACACACACACAACCTATCCCTTTCCTAGTAAAGGAGTTAATGGCACTCTGACCCTAGCTCCAAAAGGAATTTGGCAGGAGATTGGAAGGATCACGGGACAGGTTGTTTAAGGGCCAAACGGCTCT
This mutation is a 1906 bp deletion beginning at Chromosome 9 position 106,451,319 bp and ending after 106,453,224 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
45064 | GGC TGT GGG CAT GAA TGA | Common | A | |||
45065 | AAC GGC AAT GAA TGG GAA G | Wild type Reverse | A | |||
45066 | AGG AAA GGG ATA GGT TGT GTG | Mutant Reverse | A | |||
45067 | Fluorophore-1 | ACC TGG GCA CAG AGT GGA | Quencher-1 | WT Probe | ||
45068 | Fluorophore-2 | TGT TAC GTG ACA CCC ACA CAC | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
45064 | 0.50 uM |
45065 | 0.50 uM |
45066 | 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 |