Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr10:34283320+34283417 98bp TGCAATACTACCTGTGTCGTGA ACCTGACTGGAAGCCAAAAG
Mut= 99 bp
Wt= 98 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AGAAGCCAAACAAATACGGAAGTCACGAGCTGAAGAACCAATCAGGTGCGCTGCAGGTTCTCGCGAGACTTGCGGGCTGTGGCTCTGGCGTCACTGCGCTGGAGAGAGCGAGGGGCGGAGCTGGGGTTGCGGGGGACCGTGAGGGTTGTTGTTTGTAGGCGGAGGTCTGCGCGACTAGACCCGACATGAGCAGCCCGGAgcgggacgaggggacccctgtccccgacagccgcggccactgcgatgctgacactgtctcggggaccccagaccggcgaccgctgctcggcgaggagaaggcggtgacaggtgagggccgcgcagggatcgtggggagccccgcgccccgagatgtcgagggccttgtcccccagattcgggtggccgcagctaggcagggagagtcacctccttccgttcggggccccgcggcagcggtgttcgtgacaccgaaatatgtggagaaggcccaagaaactcggggagcggagtcccaggcccgtgacgtgaagacggaaccgggcaccgtcgcggctgcagcggaaaagtcagaagtggccacgccagggagcgaggaggtgatggaggtggagcagaagcccgcgggcgaagaaatggagatgctggaggcgagtggaggagtgagggaggcgccggaggaggcagggccctggcacctgggcatagatcttcgcaggaacccgctggaggccatccagctggaactggacactgtgaatgctcaggccgacagggccttccaacatctggagcagaagtttgggcgaatgcgacgacactacctggaacggaggaactacatcattcagaatatcccaggcttctggatgactgccttccggaaccaccctcagttgtccgccatgattaggggtcgggatgcagagatgttaaggtacgtaaccagtctggaggtgaaggaactgagacaccctaagactggctgcaaattcaagttcttttttcgaagaaacccctacttcagaaacaagctgattgtcaaggaatatgaagtgagatcatctggtcgagtggtgtctctttccactccaatcatttggcggagagggcatgagccccagtccttcattcgcagaaaccaagatctcatttgcagcttcttcacctggttctcggaccacagccttccagaatctgacagaatcgctgagattatcaaagaggatctgtggccaaacccactgcaatactacctgtgtcgtgaaggaatccgtagaccccgacgtcgcccaaTAAGGGAGCCAGTGGAGATTCCCAGGCCTTTTGGCTTCCAGTCAGGTTAATCTTTGCATTTGAAAATGCGGCCGGAACTGGTCCCTTCTCACCTGTGCTTGAGCAAATGCAACGGGTATGCAGTGTTTTGATTTCTTTCCTCCTGACTTTTCTGCATCTTCTATCTGGATAGT
This mutation is a 1076 bp deletion beginning at Chromosome 10 position 34,282,295 bp and ending after 34,283,370 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42902 | TGC AAT ACT ACC TGT GTC GTG A | Wild type Forward | A | |||
42903 | ACC TGA CTG GAA GCC AAA AG | Common | A | |||
42904 | GTT GTT TGT AGG CGG AGG TC | Mutant Forward | A | |||
42905 | Fluorophore-1 | CCG TAG ACC CCG ACG TC | Quencher-1 | WT Probe | ||
42906 | Fluorophore-2 | CAT GAG CAG CCC GGA TAA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42902 | 0.40 uM |
42903 | 0.40 uM |
42904 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
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 |