Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr2:30107303-30107406 104bp ACTCTCCTCGCACAGTTGCT CAGAGGTAGGATGGCCACAG
Mut= 116 bp
Wt= 104 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
AACAGAAAGAGGAGCTTATTCTTGCTTGTCAGTGAAATGTGTCTGTGGTATAATGGCAGGGGACCTTTCAAAGGTACTGGCAGTGGTGGCAGCTCCCAGTCCATGTTCTACAGTACGCCCTCCCACCCTTCcatcaagaagtgagagcagggaatgcagctcaagtggtacaaagctctgactttgatcaaagcaggcagagaagcatgtgcctgtatcccagcacttgagaggtggggtaggaggaccaggggttgaaggtcaggctcagctccacagtgggttgggcagggacaggatgagattagaagccgttgtttctctatgggtggagttcctggtccatgggggagcccaggctaggtccagtcaggtggctagctctgacatgctctttccccagcaccgagccatccaagagcagcatcatgcccttccgggctctgaagaggccactgcagttccttgggctctttgagacctcattgtgtcgtcttacgcacattccagcctacaaagtaagaggtggagttgggaggaggaagtggccaggtaccgcatagcactgagtctggggtcccattacctgggttgtcctgacatgttctgtcactaggtgagtggtgacaaaaatgaagagcaggtgctgaacgccatcgaggcctacacagagcaccggcccgagatcacatccagggccatcaacctgctgtttgacattgcacgcattgaacgctgcaaccagcttctgcgggccctgaaggtcagctcagaacccaaggccctcaggctcccagatactgctgccctccttctctgtccttctgcacccagccctgatcccaggggaacaggtagtcttgccttgcaaacctccgccctttggcagtgctggcctcactgccagcctgtccaggccctgccctagtttctgatctcacatctccccaggagagcaagtctaaggtcagcctttccccagagaagacacggtgacttaaagagcccaggctgtctttgcctgcctctggcagtgccccagctccagccccttccctgtgttcacacagctggtcatcacagccctcaagtgtcacaagtatgacaaaaacattcaagtgaccggcagtgctgccctcttctacctgaccaactccgagtaccgctcggagcagagcgtcaagttgcggcggcaggtcatccaggtggtgctgaatggcatggagtcctaccaggaggtgacggtgagcccctgcctgctgcggcctgcatgcttaggcccagtatcccttcctgctccccaggctccccaggcttagggggcctttgccaactctcctcgcacagttgctgtcccagaaacctgcccctggcacacagggaccgcagaagctggcttcAACTGCAGTCCCCTGCCTGTGGCCATCCTACCTCTGCCCTCTGTGTTCCTGATTTGCCATTTTCTCTGCCCCTCTCTCCCTATCATTGTTGGGGGTGGGGGAGTT
This mutation is a 1269 bp deletion beginning at Chromosome 2 position 30,108,607 bp and ending after 30,108,607 bp (GRCm38/mm10).
Designed assuming this was supposed to be from 30,107,338 bp and ending after 30,108,607 bp. - DSW 7/27/18
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
41674 | ACT CTC CTC GCA CAG TTG CT | Wild type Forward | A | |||
41675 | CAG AGG TAG GAT GGC CAC AG | Common | A | |||
41676 | AAT GGC AGG GGA CCT TTC | Mutant Forward | A | |||
41677 | Fluorophore-1 | CCC AGA AAC CTG CCC C | Quencher-1 | WT Probe | ||
41678 | Fluorophore-2 | CCA CCC TTC AAC TGC AGT C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
41674 | 0.40 uM |
41675 | 0.40 uM |
41676 | 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 |