Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination. The transgene genotype is determined by comparing ΔCt values of each unknown sample against known homozygous and hemizygous controls, using appropriate endogenous references.
Mutant= 79 bp
Wild Type = 94 bp
>chr5:108819412+108819505 94bp TGTGGCCTCAAAGATACATCC GGATCAATACAAACCTGCACAAC
Wt Sequence: ctacccatgtcctacagcccctctccaaggtttataGTcactctgtggcctcaaagatacatcctTGgtggcacatcatgtctactactgttatgatgattctgtgacctgcaggttgtgcaggtttgtattgatcccagactgagggaaaaggagtaaccgtcaaagtcagcaaaatccatatttccaccagtggcatcaggacactctgcatcgccaaggacagaata
Mut Sequence:
TTATAGTCACTCTGTGGCCTCAAAGATACATCCTTtgaaagacccccgaggtgggtagtcaatcaatctgaggagaccctcccaaggatcagcgagaccacgattcggatgtaaacagcaagaggctttattgggaacacgggtacccgggcgactcagtctgtcggaggactggcgcgccgagtgtggggtttttaccctttttatagggctggggagcaaaaagcgt
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
36815 | TGT GGC CTC AAA GAT ACA TCC | Common | A | |||
36816 | TGA TCC TTG GGA GGG TCT C | Mutant Reverse | A | |||
36817 | GGA TCA ATA CAA ACC TGC ACA AC | Wild type Reverse | A | |||
36818 | Fluorophore-1 | ACC CCC GAG GTG GGT AGT CA | Quencher-1 | MUT Probe | ||
36819 | Fluorophore-2 | TGG CAC ATC ATG TCT ACT ACT GTT ATG A | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
36815 | 0.40 uM |
36816 | 0.40 uM |
36817 | 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 |
---|---|
037206 | B6.129-Optntm1.1Htse/J |
004202 | B6.C3 Pde6brd1 Hps4le/+ +-Lmx1adr-8J/J |
000002 | B6.C3-Pde6brd1 Hps4le/J |
006576 | B6.FVB-Tg(GNAT2-Dta)98Wwk/J |
004462 | B6;C3-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax |
028590 | B6C3-Tg(tetO/Prnp-TARDBP*)2Vle/J |
002810 | B6CBA-Tg(HDexon1)62Gpb/1J |
006494 | B6CBA-Tg(HDexon1)62Gpb/3J |
001924 | B6EiC3Sn a/A-Ts(1716)65Dn/J |
004850 | B6EiC3Sn-Rb(12.Ts171665Dn)2Cje/CjeDnJ |
006554 | B6SJL-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax |
001957 | C3A Pde6brd1.O20/A-Prph2Rd2/J |
001979 | C3A.Cg-Pde6b+ Prph2Rd2/J |
024703 | C3A.Cg-Pde6b+Tg(Thy1-CFP)23Jrs/SjJ |
003648 | C3Sn.BLiA-Pde6b+/DnJ |
000654 | CBA/CaJ |
000656 | CBA/J |
004624 | FVB.129P2-Pde6b+ Tyrc-ch Fmr1tm1Cgr/J |
004828 | FVB.129P2-Pde6b+ Tyrc-ch/AntJ |
022880 | FVB.Cg-Pde6b+ Lrrk1tm1.1Mjff Tyrc-ch/J |
003342 | STOCK Hbatm1Paz Hbbtm1Tow Tg(HBA-HBBs)41Paz/J |
005441 | STOCK Tg(CAG-DsRed*MST)1Nagy/J |
004808 | STOCK Tg(MAPT)8cPdav Mapttm1(EGFP)Klt/J |
010815 | STOCK Tg(mI56i-flpe)39Fsh/J |
24 strains use this protocol |