Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr5:127359407+127359488 82bp GTTGGCAGTTTCCCGAAG ACGCAGTGATTTAGGACAAA
Mut= 114 bp
Wt= 82 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletions in lower case):
GTTCCTCTGTCTCTGCTCCCGCCCCACCCCCACCCAAGTGCTAGGACCACACCCATAAGCCACCATGCACTGCCTCTTTTGTTTTAGATTATGGAGATCTGAACTCAAGTCCTCAAGTTTTCATGGCAAGCACTTTACTAAGCCACCAACTCCCAGTTTTACACACTTCAATCTACTTTATATAATTAAACCTAATAATTCTGCAATTAGTGGACCACCGGGAAGAGTTGGCAGTTTCCCGAAGGACTCCGTAGctttggtcgtcgtggactttagaactcgtagctctttgtcctaaatcactgcgtgcaaactaacagcccggataatagctgcctgttactgatttaaacttcttcccctctcttccttagtgaccgaaagccgaggggtcatggataatatacagagattctcttcattgccgccgtacctgcccgtgagcttccacgtcctcagagccgagactgcattcttcctaaaggaggccaaccccgacccgctgcggaatgccagcctgcagtccagggtggagtctttcttcatctacaaggcccagcagcccccggtattaaacgtcagctatgggccttactctgcagaaaaggtcatccctctggacttgatgttgaaccccaactttttaggcccaaccagtaagtttccctttgactggaggctgaaggcctacatccttcaagagaaagtctacctgagccatcccaaagtacaggtgctcttccacatcgtgggccgagactgggatgaccacagggacgagaaactgccctgcctgcgggtctttgcgttcagagacagccgggaggttcgaggcagctgtcgtctgggtgggcccctgggactgtgcgtggcccagctggagatgctgcctggctggttcagtcccccagcggttgtatctgggcgcaggaggccagcagagcggccagaggggagtccggtggaactgtattatgctgtacagccaggggacgagcgtggtgactgcactggaggtgacaccaggaaggacaatgctattcgtccaggaaaggatggacaggagggcaggacatcccacctacagaagattggcaccattagcctttaccgcgcccaggacagcacccagctcagcgaactgcgcctggatggcaatgtggtcatctggctgccctcccagcccgtcaagcagggagacatagtcaccgcatctgtcaccatcgccaataactctactgtggaccatttcatcctaaggtaggcacccaggctggtatctgattgtatgtagtatgtctagggtcaatctgactcctcgcctaagggtcggcgtaattgctacctcccaagGGATCTGCCCCCTAATACAGAAACCAGTGATTCCAAGCCAAGATGCGCTGGAAAGCTGTGCTTAGGCTGTGTTTGTTGTGGGCGATGAACGGGAAGAGGCGAGTTAGTAGGTGGCCTCTTTCTCTCTATCAGCTACAGAATTAAAATTCAGGAAAACCACCGAGCAAGATCAATGCCTTTACTTGCAAATGTTATAGACTTCTCAGATAAAGAAAGGCTCCCCAGAACTGGGGAGACCACCCACTTTAAGAGGCTTGGGGTTTTTTGTTTTGTTTTGTTTTGTTTTGTTTGT
This mutation is a 1,099 bp. deletion of Chr5:127,359,435-127,360,533 (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
73352 | GTT GGC AGT TTC CCG AAG | Common | A | |||
73353 | ACG CAG TGA TTT AGG ACA AA | Wild type Reverse | A | |||
73354 | ATC GCC CAC AAC AAA CAC | Mutant Reverse | A | |||
73355 | Fluorophore-1 | CGT CGT GGA CTT TAG AAC TCG | Quencher-1 | WT Probe | ||
73356 | Fluorophore-2 | ATT CCA AGC CAA GAT GCG CT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
73352 | 0.40 uM |
73353 | 0.40 uM |
73354 | 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 |
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