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Stock No: 013087
Protocol 29681: Standard PCR Assay - Generic LacZ Melt Curve Analysis
Version 4.2
Notes
The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX).
To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility.
Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.
Expected Results
Transgene Tm = 87°C +/- 1.0°C
Internal positive control Tm = 82.1°C+/- 1.0°C
Internal positive control Tm = 82.1°C+/- 1.0°C
JAX Protocol
Protocol Primers
| Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
|---|---|---|---|---|---|---|
| oIMR0040 | CGT GGC CTG ATT CAT TCC | Mutant Reverse | A | |||
| oIMR3054 | ATC CTC TGC ATG GTC AGG TC | Mutant Forward | A | |||
| oIMR8744 | CAA ATG TTG CTT GTC TGG TG | Internal Positive Control Forward | A | |||
| oIMR8745 | GTC AGT CGA GTG CAC AGT TT | Internal Positive Control Reverse | A |
Reaction A
| Component | Final Concentration |
|---|---|
| ddH2O | |
| Kapa 2G HS buffer | 1.30 X |
| MgCl2 | 2.60 mM |
| dNTP KAPA | 0.26 mM |
| oIMR0040 | 0.50 uM |
| oIMR3054 | 0.50 uM |
| oIMR8744 | 0.50 uM |
| oIMR8745 | 0.50 uM |
| Glycerol | 6.50 % |
| Dye | 1.00 X |
| Kapa 2G HS taq polym | 0.03 U/ul |
| DNA |
Cycling
| 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 |
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.
Strains Using This Protocol
| Stock Number | Strain Name |
|---|---|
| 012668 | 129S.129-Lrp5tm1Grw/J |
| 038651 | 129S1.Cg-Tg(Hmgcr-lacZ)H253Sest/EmsJ |
| 002957 | B6.129-Dll1tm1Gos/J |
| 004276 | B6.129-Figntm1Frk/Frk |
| 004478 | B6.129-Foxd1tm1Lai/J |
| 003870 | B6.129-Plin1tm1Chan/J |
| 019146 | B6.129-Tmc1tm1.1Ajg/J |
| 030985 | B6.129P2(Cg)-Aph1atm1Bdes Aph1btm1Bdes Aph1ctm1Bdes/J |
| 009348 | B6.129P2(Cg)-Hprt1tm17(Ple48-lacZ)Ems/Mmjax |
| 012575 | B6.129P2(Cg)-Hprt1tm39(Ple24-lacZ)Ems/Mmjax |
| 012333 | B6.129P2(Cg)-Hprt1tm45(Ple67-lacZ)Ems/Mmjax |
| 012733 | B6.129P2(Cg)-Hprt1tm53(CAG-lacZ)Ems/Mmjax |
| 012347 | B6.129P2(Cg)-Hprt1tm64(Ple170-lacZ)Ems/Mmjax |
| 012583 | B6.129P2(Cg)-Hprt1tm68(Ple127-lacZ)Ems/Mmjax |
| 012656 | B6.129P2(Cg)-Hprt1tm70(Ple240-lacZ)Ems/Mmjax |
| 012657 | B6.129P2(Cg)-Hprt1tm71(Ple155-lacZ)Ems/Mmjax |
| 012659 | B6.129P2(Cg)-Hprt1tm73(Ple142-lacZ)Ems/Mmjax |
| 012734 | B6.129P2(Cg)-Hprt1tm74(Ple232-lacZ)Ems/Mmjax |
| 018054 | B6.129P2(Cg)-Pitx2tm1.1Dmm/J |
| 013144 | B6.129P2-Aim2Gt(CSG445)Byg/J |
| 005801 | B6.129P2-Esrratm1Dgen/J |
| 005814 | B6.129P2-Grm1tm1Dgen/J |
| 034597 | B6.129P2-Kif13aGt(AF0553)Wtsi/AhcJ |
| 023209 | B6.129S-Sfrp1tm1Jrbn/PcmaJ |
| 004950 | B6.129S1-Syt7tm1Nan/J |
| 036325 | B6.129S6-Mecomtm1Aspe/Mmjax |
| 005876 | B6.CAST-(rs4222996-rs13476494)/LusJ |
| 019547 | B6.Cg-Cacna1ctm2Itl/J |
| 017762 | B6.Cg-Cacna1ftm1.2Sdie/J |
| 030065 | B6.Cg-Serpinf1tm1Craw/J |
| 006477 | B6.Cg-Tg(CAG-lacZ-WGA)330Bbm/J |
| 033345 | B6.Cg-Tg(Cdh5-COP4*L132C,-lacZ)B28-2Mik/J |
| 032889 | B6.Cg-Tg(Cdh5-RHO/Adrb2,-lacZ)B27-3Mik/J |
| 006229 | B6.Cg-Tg(DRE-lacZ)2Gswz/J |
| 024050 | B6.Cg-Tg(Hmgcr-lacZ)H253Sest/J |
| 023757 | B6.Cg-Tg(tetO-tetX,lacZ)1Gogo/UmriJ |
| 028353 | B6.Cg-Tg(Vil1-EGFP)106Gum/J |
| 018625 | B6.FVB-Tg(Fabp4-lacZ)4Mosh/J |
| 025740 | B6;129-Prg4tm2Mawa/J |
| 008704 | B6;129-Smn1tm6(SMN2)Mrph/J |
| 025197 | B6;129-Vmn1r51tm1Dlc/J |
| 025196 | B6;129-Vmn1r51tm2.1Dlc/J |
| 017524 | B6;129-Tg(CMV-Bgeo,-WGA,-ALPP)1Mgmj/J |
| 038621 | B6;129P2-Ctcftm2.2Gal/J |
| 024288 | B6;129P2-Faptm2Schn/J |
| 006744 | B6;129P2-Gt(ROSA)26Sortm1Mom/MomJ |
| 006665 | B6;129P2-Or8a1tm13(Olr226)Mom/MomJ |
| 012850 | B6;129P2-TardbpGt(RRB030)Byg/J |
| 026012 | B6;129P2-Uts2rtm1Djbe/J |
| 006465 | B6;CBA-Tg(CAG-lacZ-WGA)330Bbm/J |
| 007972 | B6;CBA-Tg(Olfr151-taulacZ)4Mom/MomJ |
| 006680 | B6;CBA-Tg(Olfr16*,taulacZ)19Mom/MomJ |
| 006672 | B6;CBA-Tg(Olfr16*,taulacZ)7Mom/MomJ |
| 007973 | B6;CBA-Tg(Olfr16-taulacZ)1Mom/MomJ |
| 007974 | B6;CBA-Tg(Olfr160-taulacZ)V4-7Mom/MomJ |
| 007976 | B6;CBA-Tg(Olfr713-taulacZ)4Mom/MomJ |
| 007975 | B6;CBA-Tg(OR8A1-taulacZ)1Mom/MomJ |
| 004141 | B6;CBA-Tg(UAS-lacZ)65Rth/J |
| 008344 | B6;DBA-Tg(Fos-tTA,Fos-EGFP*)1Mmay Tg(tetO-lacZ,tTA*)1Mmay/J |
| 018627 | B6;SJL-Tg(Myl1-lacZ)1Ibdml/J |
| 002621 | B6;SJL-Tg(tetop-lacZ)2Mam/J |
| 003299 | B6;SWJ-Tg(TIMP3-lacZ)7Jeb/J |
| 037014 | B6J.129P2-Pofut2Gt(RST434)Byg/BchMmjax |
| 024866 | B6J.B6N(Cg)-Sec24ctm1a(EUCOMM)Wtsi/J |
| 034731 | B6N(129S4)-Srd5a3tm1c(EUCOMM)Wtsi/J |
| 030130 | B6N.129-Dkk2tm1Dwu/J |
| 030043 | B6N;129-LrbaGt(OST69142)Lex/WgkJ |
| 002754 | C57BL/6-Tg(LacZpl)60Vij/J |
| 023048 | C57BL/6-Tg(Peg3-lacZ)26Sass/J |
| 027898 | C57BL/6J-Tg(CAG-lacZ,-FUS,-EGFP)629Gyu/J |
| 037062 | FVB.129S-Neu1tm1Adz/J |
| 005025 | FVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg(SMN2*delta7)4299Ahmb/J |
| 026047 | FVB/N-Tg(Isl1-lacZ)5Cka/Mmjax |
| 002856 | FVB/N-Tg(TIE2-lacZ)182Sato/J |
| 035185 | STOCK Adgrl1tm2c(EUCOMM)Hmgu/SudJ |
| 026229 | STOCK Akap12tm1Ihg Rb1tm2Brn Tg(Pbsn-cre)4Prb/J |
| 035392 | STOCK Gsctm2Bhr/J |
| 024284 | STOCK Hcn4tm3Sev/J |
| 013764 | STOCK Hprt1tm57(Ple26-lacZ)Ems/Mmjax |
| 012354 | STOCK Hprt1tm66(Ple5-lacZ)Ems/Mmjax |
| 012584 | STOCK Hprt1tm69(Ple134-lacZ)Ems/Mmjax |
| 013087 | STOCK Mir7-2tm1Mtm/Mmjax |
| 006578 | STOCK Myoz2tm1Eno/J |
| 006686 | STOCK Or6b9tm1Mom/MomJ |
| 006613 | STOCK Tg(CAG-Bgeo,-Tle1,-ALPP)1Lbe/J |
| 004623 | STOCK Tg(TCF/Lef1-lacZ)34Efu/J |
| 005728 | STOCK Tg(tetO-Ipf1,lacZ)958.1Macd/J |
| 87 strains use this protocol | |