Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr6:127967488-127967586 99bp TCTGCCCAGAGGAGTAACCT TCATGAGGTCTCTCTGAAGATCA
Mut= 121 bp
Wt= 99 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
ACCCATGGACTTGCTTTCCAGACTTCAGAAAACAGGGGTTACGTGACCCTTCTGTTCAGGGTAGTGGGAAACACCCACTGAGGTTTTGGCTTTGAGAGCCAGTGACCTGGGTTCTCCCCCGTAGACCAACCAGTCATACCCAGACCCAGCCTCCCTTCTGAGCATGGGGTCCTCTCTGCCCAGAGGAGTAACCTGACACACATTTGTGTAGTTTTCTGGTTTCCCCcttcgtctcactgaacactgaagttgatcttcagagagacctcatgaggtatatgaagctgcctcgtcccatttactgtaggggagaccgaggcatgcagtgacaagggggcatgtcataggctttgggtctgtgacagcgcctggctttttataatcattttgtatctcagagtcagtggtgagagacatgcatggggtggggtggtctgggagatgaatgtaagagctgtgctggtctaaccagtttcttctttctccagctctgtggctgtgggctgcttggagtgggcatctggctctctgtgtcccaaggcaacttcgccaccttctcccccagcttcccctcactgtccgcagccaacctcgtcatcgccatcggcaccatcgtcatggtgacgggcttcctgggctgcctgggggccatcaaggagaacaagtgtctgcttctcagcgtaagtcggccctcgcgttcccttcgttgctctctggggctccctagctgaagtccacagacattgcttctagagcgttctgtccccagcccttcctgtccccctctctctttgacacatgcctgggtttgcttaggagtgggtggtgcccacttccaggctcccagcctggaaaccctgcccatcccttatctcccctcccctgccccattctctgtcaccacccaccccggccatctccactcccctgggcccacgcttgcacttgtctttccagttcttcatcgtgctgttgatcatcctgctggcagagttgattctgatcatcctcttcttcgtctacatggacaaggtaaacctcgcagaggcaggcaagatggaaggtcgccatcttgacaccagggctgggacaggggaacgcctggctaacagatcctgcagactggtctgcaggaggcctgagggtcccaccctagccacttcccctccccgtctggaaacactgctccagcattcccagctggcccagtctgggttagggaggagggccagtggcctggtctcagctgacctgtggtgtttcccctcatctgccaggtgaacgagaatgccaagcaggacctgaaagaggggctgctgctctacaacacagagaacaacgtggggctcaagaacgcctggaacatcatccaggccgaggtgcggctccagtctgtgcatggcgtaggatggggcagccatagtgcacctgtgctgttgggagaaccacctgcactggacacttgcctagacctgaagggagatgctagcatgggatgctgccggggaggggtaaagaggtgggcgcacgaggcacatgatgcgtgtctcaggagtgggcgagcctaacgtggtgaatgacaggagtcaccagatcacgtttggctcaggtgacaggctctggtagtgacaggaatacaagtctctgttgcccgatgcacaccccacatagaccctgccaatcagcttgggagggcccgtactacaaaggaaggaggtcaaggcactggtgagcaagtattgggctgggctctttaactccacatctggtgctcagcccagagggacagcagcctttctggtgtaggaaagaagagtcactcactacctctgggggcccggctgcagctcaagaggggacaaggagacattcttttccctatccctgtgttcttgggtggggttgacgtatctggccttgtgccccacagatgcggtgttgtggggtcactgactacacagactggtatccggtgctcggggagaacacagttcctgacagatgctgcatggagaattcccagggctgtgggcgcaacagcaccacccccttgtggagaacggtgagactcactgatcccctgtgcgctggcagcaggaatgatgggaattagtgcagtgtgggatggagagagggctacactggagtgaagccagcagtgctgtgtgtgtgtgtgtgtgtgtgtacacatgcacgtatgtgcaggactcacacttgggattcagaaggtaaggggttggatggggattaggcagtgcagctgcctgctgtaagtggagcctgggggttggatgttgcttctggggagggccagggcctaagggtagtaacggactagaattcagaataaagacaggggctagggcaaaatgacggaagggttgggatggccccaaccagtgtcgtgccaggaccttaccctgtggttcacccccccagggctgctacgaaaaagtgaagctgtggtttgatgacaacaagcacgtgctaggcaccgtgggcatgtgcatcctcatcatgcaggtgaggggaccccgggcacctcgactctgggtggacgcaagccagagccaccgccaggctgcagggctccgagtgaggccacaggcaatgggcaaaggtggtgctcagtgctaggcggggccctgacatgctccgcatgagagctaaaggtgttcagtgtcccccgggtcaccagCTTTACAGGTGACATGTGGCACCTTGGGTGAAGCCACAGGTGGCCCCTGCCTGCACCTCTGTGTCTCCACctgctttcAGGGTTTTAGCTGAGGAGCGTAAATGGGATTTGATGTTGTTCAAGACAGTTATCCAAGACATCACTTCACGTGTTCTAGAACCACAGAGCTTGTTGGCATGAATGCCAATGACTCGAACACCCCCTTG
This mutation is a 2472 bp deletion beginning at Chromosome 6 position 127,965,063 bp and ending after 127,967,534 bp (GRCm38/mm10). In addition there is an 8 bp deletion (CTGCTTTC) 70 bp after the 2472 bp deletion that will not affect the results of the deletion.
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
39262 | TCT GCC CAG AGG AGT AAC CT | Common | A | |||
39263 | TCA TGA GGT CTC TCT GAA GAT CA | Wild type Reverse | A | |||
39264 | TGG AGA CAC AGA GGT GCA G | Mutant Reverse | A | |||
39265 | Fluorophore-1 | CTT CGT CTC ACT GAA CAC TGA AGT | Quencher-1 | WT Probe | ||
39266 | Fluorophore-2 | TTC TGG TTT CCC CCT TTA CAG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
39262 | 0.40 uM |
39263 | 0.40 uM |
39264 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
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 |