Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr15:81388589-81388683 95bp TGCCATCTCTCCAGCTTACC AAGCTAGTTTCACCTAAACCCTGA
Mutant= 111 bp
Wild Type = 95 bp
Wt Sequence (deletions in lower case):
ATGTATGTGGTACTTTCAGAAGCCAGATCCCCTGGTACTGGAGTTGCCATCTCTCCAGCTTACCTAGTAATAGCTAGTAGTAGTTATTTctagcagggaagtagttaataatgtatcagggtttaggtgaaactagcttatagttctactagcaagtagttcattttgaggaagggatatacctttaaccttcatcctattggatagtacttagccctgagctccacacaaatcagttcatctcattttacagtttcaagccagtggctgttaactgtaccacactcgtaatttaggtttcaatctcatggttttcaccttttgcagAAATTGACAATGAAGGTGTAATTGAACCAGACACTGATGCCCCTCAGGAAATGGGAGATGAAAATGCAGAGgtaagtaagtgacattttaggaaaagtgaagttccccggggttggattattttataatataactcccaagatgtgtatgataaatcttcctttgactgatgacatactttataatttCACTAAAAGATTCAAAAATTCCCATGTTGTTTTTAACTATTATCTCTGACGTCTTGTATTTTGTTTTACTTGTTCTATCAATTTACTTCATTTTGACTAACCTGTAAAATTATTTCTCTAAAATAGATACCTACAATTAGAATTGTTCTTTTTTTTTTTTTTTTGAGATGATTTTGTCTTTTTTTCAAGAC
This is a 426 bp deletion beginning at Chromosome 15 position 81,388,213 bp and ending after 81,388,638 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
47603 | TGC CAT CTC TCC AGC TTA CC | Common | A | |||
47604 | AAG CTA GTT TCA CCT AAA CCC TGA | Wild type Reverse | A | |||
47605 | AAA CAA AAT ACA AGA CGT CAG AGA | Mutant Reverse | A | |||
47606 | Fluorophore-1 | TAT TTC TAG CAG GGA AGT AGT TAA TAA TG | Quencher-1 | WT Probe | ||
47607 | Fluorophore-2 | AGC TAG TAG TAG TTA TTT CAC TAA AAG ATT CAA | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
47603 | 0.40 uM |
47604 | 0.40 uM |
47605 | 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 |