Stock No: 025740
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

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
017762 B6(Cg)-Cacna1ftm1.2Sdie/J
002957 B6.129-Dll1tm1Gos/J
004276 B6.129-Figntm1Frk/Frk
004478 B6.129-Foxd1tm1Lai/J
019146 B6.129-Tmc1tm1.1Ajg/J
030985 B6.129P2(Cg)-Aph1atm1Bdes Aph1btm1Bdes Aph1ctm1Bdes/J
009348 B6.129P2(Cg)-Hprttm17(Ple48-lacZ)Ems/Mmjax
012575 B6.129P2(Cg)-Hprttm39(Ple24-lacZ)Ems/Mmjax
012333 B6.129P2(Cg)-Hprttm45(Ple67-lacZ)Ems/Mmjax
012733 B6.129P2(Cg)-Hprttm53(CAG-lacZ)Ems/Mmjax
012347 B6.129P2(Cg)-Hprttm64(Ple170-lacZ)Ems/Mmjax
012583 B6.129P2(Cg)-Hprttm68(Ple127-lacZ)Ems/Mmjax
012656 B6.129P2(Cg)-Hprttm70(Ple240-lacZ)Ems/Mmjax
012657 B6.129P2(Cg)-Hprttm71(Ple155-lacZ)Ems/Mmjax
012659 B6.129P2(Cg)-Hprttm73(Ple142-lacZ)Ems/Mmjax
012734 B6.129P2(Cg)-Hprttm74(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
005876 B6.CAST-(rs4222996-rs13476494)/LusJ
019547 B6.Cg-Cacna1ctm2Itl/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
006665 B6;129P2-Olfr151tm13(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
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
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
023048 C57BL/6-Tg(Peg3-lacZ)26Sass/J
027898 C57BL/6J-Tg(CAG-lacZ,-FUS,-EGFP)629Gyu/J
008604 FVB.129(B6)-Smn1tm5(Smn1/SMN2)Mrph/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
024284 STOCK Hcn4tm3Sev/J
013764 STOCK Hprttm57(Ple26-lacZ)Ems/Mmjax
012354 STOCK Hprttm66(Ple5-lacZ)Ems/Mmjax
012584 STOCK Hprttm69(Ple134-lacZ)Ems/Mmjax
013087 STOCK Mir7-2tm1Mtm/Mmjax
006578 STOCK Myoz2tm1Eno/J
006686 STOCK Olfr6tm1Mom/MomJ
006613 STOCK Tg(CAG-Bgeo,-Tle1,-ALPP)1Lbe/J
005493 STOCK Tg(Tek-rtTA,TRE-lacZ)1425Tpr/J
005728 STOCK Tg(tetO-Ipf1,lacZ)958.1Macd/J
75 strains use this protocol