Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
>chr3:106733028+106733143 116bp ACAGAATGCCCCCAGAAAAG CCTGTGATTCTTGGTCTCTGA
Mut= 124 bp
Wt= 116 bp
Fam=Mut
Hex=Wt
Wt Sequence (deletion in lower case):
TTGCCTCCTTTGGTTCAGCAAAAGATACTTGGAACTGCAACCACAAGTACCTCAGGAACAGTTGAGGCCTCCCAAATACCAACAGTTGTTTATGTACATCCTGTAAATTCAGTGAAATTTGTTGTTACCAAGAAAACTCAGACTATTTACCCCAAACCAGTGACATTCAATACCTTGCAAATTCCACCAAATGTTGCTACAGAAACACAATTAAAAGGTGGTCAGCATCCTCAAGCTGCTCCAGTGAACTCGatttttcaggagtatctacagcctggaatcccatgtattattcctgttaagtcttcaaataatgtggctacaaaggttttaaacacttttgtaggtaggaaaaatctgggagataatacaatagatacacctctgttgaataccaacagttctggcaggacacactccgtaagtgaacctattaaagataatgctttgataatgtttaatgggaaagtctggttgaatgaaaaggggacatgcggtctgccatcaaaaattgaccagcagaattctgtttcttctgatattccactaaaagattcatcacagctagtaagttcaagtatagtcacagaaatatccagagagattttaaatagtgttttggttaaaagtaaatcttttcagttgaagacaaaatcactttcaaattcccagcttgcttccatggccaatctaagagcagagaagaatgaaaaagtggagagaccatctttttctgtcacaaatccacatactatgaaccagtccactcactgccttaaacagagtaagactgtatttataaacccagtttttccagatggatttaggacaggacagaatgcccccagaaaaggaaatctcgtccagaacatagagaaaatatgttcctctgttgATGCAGCAACTGTTACTTCTCAACAGTGTGTTTTCAGAGACCAAGAATCACAGGTAATTTTCCCACATCAGGATGCTCTTTATATATATATATATATGTGGAAACTATTTCTTAATTCTCAAAATTATCTGATACCTGTCTCCTACACACACACCCCATCACAAGGCTTGCTTAAAATTTTTATCTGAATAAATATTTGTGTACAAGTAGTAGAGCCTAAGCAATATCTTTCTCTTTCAT
This mutation is a 643 bp deletion beginning at Chromosome 3 position 106,732,447 bp and ending after 106,733,089 bp (GRCm38/mm10).
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
49000 | ACA GAA TGC CCC CAG AAA AG | Wild type Forward | A | |||
49001 | CCT GTG ATT CTT GGT CTC TGA | Common | A | |||
49002 | TCC ACC AAA TGT TGC TAC AGA | Mutant Forward | A | |||
49003 | Fluorophore-1 | CCA GAA CAT AGA GAA AAT ATG TTC CTC | Quencher-1 | WT Probe | ||
49004 | Fluorophore-2 | CCA GTG AAC TCG ATG CAG C | Quencher-2 | MUT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
49000 | 0.40 uM |
49001 | 0.40 uM |
49002 | 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 |