Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr19:22711543+22711667 125bp AAACTCGAACCCCTTTATCTCC AGGCTGCTTGTCATTGTGA
Mut= 115 bp
Wt= 125 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
GTTTGTTTAAACTACATTTAATAAAAATCAAGTAATTTCAAAATTGCCAATAACTTAGAATTTCAACAAAACCTTTAAAACTCGAACCCCTTTATCTCCTTTTCTCTAAAGGAAATGGGCCCTTTTCCTTACatttctgatttcacataagacattttcttcaagtagggacacatataactgtcacaatgacaagcagcctcaggcagctttgcccatctccctaattgtcaatctctattctagtatgtccgagtatcttttgatacgaaacctgatctcttgctacacctgatgaccaaggaatggcaactggagcttcccaaacttctcatctccgtgcacggagggctgcagaactttgaactccagcccaaactcaagcaagtcttcgggaaggggctcatcaaagcagccatgacaactggagcctggatcttcactggaggggtcaacacaggtaatcagagtaggatggagaaaggcagctgcaaccacattgtatccagaataattttgcctcaggttgaaaacgcccatacttctctacctcacctctACTCATATGTGCCCGCCCTCCTGCTCACCAACCTCCTCTTATTGCTTCTTCTCCTTCCTGTGCCTTAGTGAGAGCTATGCAGATATGTTTCAGCTAAGAAGAACTGCATATGAGGAATGG
This mutation is a 427 bp deletion beginning at Chromosome 19 position 22,711,598 bp and ending after 22,712,024 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42939 | AAA CTC GAA CCC CTT TAT CTC C | Common | A | |||
42940 | AGG CTG CTT GTC ATT GTG A | Wild type Reverse | A | |||
42941 | CAG GAA GGA GAA GAA GCA ATA AG | Mutant Reverse | A | |||
42942 | Fluorophore-1 | AAG ACA TTT TCT TCA AGT AGG GAC AC | Quencher-1 | WT Probe | ||
42943 | Fluorophore-2 | CCT TAC ACT CAT ATG TGC CCG | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42939 | 0.40 uM |
42940 | 0.40 uM |
42941 | 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 |