Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 115 bp
Wild Type = 135 bp
>chr19:24351641-24351775 135bp TGGCTTCTAGCATTTGAACCT TGCCTGAGGTAGAAGGTGGT
Wt Sequence:gggggtagacgtggggatcagtccctcagcactgtacctgagatgctgtaattggcTActctcgagtataggagagccaatctataaaccatgtattgctggatcagagtagacgaagatgtggtaattagtgtttttaactttggcttctagCATTTGAACCTCCACTACCTCCAGATTCTGAACATCAAAAAGCAGAGCGTCTGCGTGGTGCATTTGAGGAACTTGGGGACATTGGACAACCCAAGgtgagatgaaaccaccttctacctcaggcatgccttccacttcctctctcctcatatggaaaccatttctccctgggaacaaccttgagtgacgtctttcacatcactaagcatcccccaTGttaccttaggtgaatggtgttactgtgtgactatcccaggcttcctttagttcattcctatggttggatatt
Mutant Sequence: TAGCAGGAAGTCAGGGAGGACCCAGAGCAAAACTTTGGGGGTAGACGTGGGGATCAGTCCCTCAGCACTGTACCTGAGATGCTGTAATTGGCTcattaatccataacttcgtataatgtatgctatacgaagttatccagGTTACCTTAGGTGAATGGTGTTACTGTGTGACTATCCCAGGCTTCCTTTAGTTCAT
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
36836 | GTC CCT CAG CAC TGT ACC TG | Mutant Forward | A | |||
36837 | TCA CAC AGT AAC ACC ATT CAC CT | Mutant Reverse | A | |||
36839 | Fluorophore-1 | TGC TGT AAT TGG CTC ATT AAT CCA TAA C | Quencher-1 | MUT Probe | ||
37347 | TGG CTT CTA GCA TTT GAA CCT | Wild type Forward | A | |||
37348 | TGC CTG AGG TAG AAG GTG GT | Wild type Reverse | A | |||
37349 | Fluorophore-2 | CTT GGG GAC ATT GGA CAA CCC | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
36836 | 0.40 uM |
36837 | 0.40 uM |
37347 | 0.40 uM |
37348 | 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 |
Stock Number | Strain Name |
---|---|
029100 | B6.Cg-Fxnem2.1Lutzy Tg(Ckmm-cre)5Khn/J |
030218 | B6.Cg-Pvalbtm1(cre)Arbr Fxnem2.1Lutzy/J |
029721 | B6.Cg-Pvalbtm1(cre)Arbr Fxnem2Lutzy Fxnem2.1Lutzy/J |
031007 | STOCK Fxnem2.1Lutzy Tg(FXN)Y47Pook/J |
030395 | STOCK Fxnem2.1Lutzy Tg(FXN)YG8Pook/800J |
030930 | STOCK Fxnem2.1Lutzy Tg(FXN)YG8Pook/J |
6 strains use this protocol |