Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr11:69879468-69879560 93bp CCCTGAAGACCTTCTGAACTC CTGACTCATCCCCCTCCTCT
Mut= 84 bp
Wt= 93 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TCTGAACGTTACCACCCAATTCTTTCCAAACCCCCAAATATCTGCTTTTTCCTTCTCCTTGCCTGTCTCTTATATTACCCAAAATTTCCCCCCCTCCTGTGTCCTCTTTACCCCCTTCCTTTGGGGGTCCCGTGACCCTAAATGTGGGGGGAACACTATATTCCACTACACTGGAGACCCTGACTCGCTTCCCAGATTCCATGCTGGGGGCCatgtttagggctgacaccctaatgccagccaaccttaacccgcaaggagatggccattacttcatcgacagggatggcaaggctttccggcacatcctcaattttttgcggctaggccgtctggacctgccccgtgggtacggagaaactgcccttcttaaggcagaggctgacttctaccagatccggcccctcctggatgccctgcgggaattggaagcctctcggggtacacctgcatccacagccgccctactccatgcagatgtagatgtcagcccccgccaggtgcacttctccgctcgaaggggcccccaccactatgagctgagctctgtccaggtggacaccttccgagccaacctcttctgcactgaccctgagtgtctggctgccatgcgcaacagatttggtgtggccattggggacagggcagaaggaggtccacattttcggctagagtgggcctcccgcccccaggaactccctgaagtagagtatcaaagactggggctgcagccactgtggactggggggccagaagatcgtcgggaggtagcgaacacgcctacattcctggaggaggtgctacgggtggctctggaacatggcttccgcctggactctgtcttcccagaccctgaagaccttctgaactctagatccttgcgctttgtgcgccactgagggtgctgccCTCCACTTGACTGCAGAGGAGGGGGATGAGTCAGCCAAGGGGCCTGTGGCTCTTCTAAAACCTCTTTCTAGTACTGGTGTGGTAGCTATGAAAGTCAGGGATTCCACCATACCTGGCCAGAGCCTAGAGCTGGGGTTCCCAGATCGAGCTGCCCCCTTCCACCCCACAAGACTCACGAATGGT
This mutation is a 697 bp deletion beginning at Chromosome 11 position 69,879,502 bp and ending after 69,880,198 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
42867 | CCC TGA AGA CCT TCT GAA CTC | Wild type Forward | A | |||
42868 | CTG ACT CAT CCC CCT CCT CT | Common | A | |||
42869 | CCA CTA CAC TGG AGA CCC TGA | Mutant Forward | A | |||
42870 | Fluorophore-1 | CGC TTT GTG CGC CAC T | Quencher-1 | WT Probe | ||
42871 | Fluorophore-2 | TGG GGG CCC TCC ACT | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
42867 | 0.40 uM |
42868 | 0.40 uM |
42869 | 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 |