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Melting curve analysis is done using a Roche Light Cycler 480.
This assay will NOT distinguish hemizygous from homozygous transgenic animals.
Transgene = ~450 bp
Internal positive control = 297 bp
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
oIMR8746 | CGC TGT GGG GCA TTT TAC TTT AG | Transgene Forward | A | |||
oIMR8747 | CAT GTC CAG ATC GAA ATC GTC | Transgene Reverse | A | |||
oIMR9020 | AAG GGA GCT GCA GTG GAG TA | Wild type Forward | A | |||
oIMR9021 | CCG AAA ATC TGT GGG AAG TC | Wild type Reverse | A |
Component | Final Concentration |
---|---|
ddH2O | |
Kapa 2G HS buffer | 1.30 X |
MgCl2 | 2.60 mM |
dNTP KAPA | 0.26 mM |
oIMR8746 | 0.50 uM |
oIMR8747 | 0.50 uM |
oIMR9020 | 0.50 uM |
oIMR9021 | 0.50 uM |
Glycerol | 6.50 % |
Dye | 1.00 X |
Kapa 2G HS taq polym | 0.03 U/ul |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 94.0 | -- | |
2 | 94.0 | -- | |
3 | 65.0 | -- | -0.5 C per cycle decrease |
4 | 68.0 | -- | |
5 | -- | repeat steps 2-4 for 10 cycles (Touchdown) | |
6 | 94.0 | -- | |
7 | 60.0 | -- | |
8 | 72.0 | -- | |
9 | -- | repeat steps 6-8 for 28 cycles | |
10 | 72.0 | -- | |
11 | 10.0 | -- | hold |
Stock Number | Strain Name |
---|---|
016198 | 129S6.Cg-Tg(Camk2a-tTA)1Mmay/JlwsJ |
003563 | B6.Cg-Tg(Cebpb-tTA)5Bjd/J |
003767 | B6.Cg-Tg(Eno2tTA)5021Nes/J |
003763 | B6.Cg-Tg(Eno2tTA)5030Nes/J |
005964 | B6.Cg-Tg(GFAP-tTA)110Pop/J |
002618 | B6.Cg-Tg(MMTVtTA)1Mam/J |
006232 | B6.Cg-Tg(Scgb1a1-rtTA)1Jaw/J |
034533 | B6.FVB-Tg(EmuSR-tTa)83Bop/DwfJ |
012433 | B6;C3-Tg(ACTA1-rtTA,tetO-cre)102Monk/J |
007678 | B6;SJL-Tg(KRT14-rtTA)208Jek/J |
006242 | C.Cg-Tg(Scgb1a1-rtTA)1Jaw/J |
006245 | C.Cg-Tg(SFTPC-rtTA)5Jaw/J |
013585 | FVB-Tg(Cdh5-tTA)D5Lbjn/J |
005625 | FVB-Tg(Pcp2-tTA)3Horr/J |
006222 | FVB.Cg-Tg(Scgb1a1-rtTA)1Jaw/J |
006225 | FVB.Cg-Tg(SFTPC-rtTA)5Jaw/J |
006209 | FVB.Cg-Tg(Tal1-tTA)19Dgt/J |
034802 | FVB/N-Tg(MMTV-tTA)25754Kuw/Mmjax |
004127 | FVB/N-Tg(Nes-rtTA)306Rvs/J |
005942 | FVB/N-Tg(Pf4-tTA/VP16)42Kra/J |
006875 | FVB/N-Tg(Tagln-rtTA)E1Jwst/J |
004937 | NOD.Cg-Tg(Ins2-tTA)1Doi/DoiJ |
005734 | NOD/Lt-Tg(Ins2-rtTA)1Ach/AchJ |
006999 | STOCK Dbttm1Geh Tg(Cebpb-tTA)5Bjd Tg(tetO-DBT)A1Geh/J |
024854 | STOCK Tg(Camk2a-tTA)1Mmay Fgf14Tg(tetO-MAPT*P301L)4510Kha/J |
003272 | STOCK Tg(Cebpb-tTA)5Bjd/J |
003273 | STOCK Tg(CMV-rtTA)4Bjd/J |
003271 | STOCK Tg(CMV-tTA)3Bjd/J |
005493 | STOCK Tg(Tek-rtTA,TRE-lacZ)1425Tpr/J |
008790 | STOCK Tg(tetO-DISC1*)1001Plet/J |
30 strains use this protocol |