Master Protocol Details





Allele:	Generic Neo
Protocol Name:	Generic Neo Melt Curve Analysis
Method:	Robotic MCA
Version:	2.0
Created:	21-September-2010	Updated:	05-June -2011



Notes:	Melting curve analysis is performed using a Roche Light Cycler 480.
Expected Results:	Mutant Tm = 90°C +/- 1.0°C Wild type Tm = 82°C+/- 1.0°C

Gel Image

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Protocol Primers

Primer	5' Label	Sequence 5' --> 3'	3' Label	Primer Type
oIMR6916	-	CTT GGG TGG AGA GGC TAT TC	-	-
oIMR6917	-	AGG TGA GAT GAC AGG AGA TC	-	-
oIMR8744	-	CAA ATG TTG CTT GTC TGG TG	-	-
oIMR8745	-	GTC AGT CGA GTG CAC AGT TT	-	-

Reaction/Components

Reaction Component	Volume (µl)	Final Concentration	Total Volume (µl)
ddH2O	3.88	-	3.88
2 X SYBR Green Master Mix (Roche)	6.00	1.00 X	6.00
DNA	2.00	-	2.00
100 X PM 85	0.12	1.00 X	0.12

Cycling

Step #	Temp °C	Time	Note
1	94	5 min	NEOTD
2	94	20 sec	-
3	64	30 sec*	* -0.5 C per cycle
4	72	35 sec	repeat steps 2-4 for 12 cycles
5	94	20 sec	-
6	58	30 sec	-
7	72	35 sec	repeat steps 5-7 for 25 cycles
8	72	2 min	-
9	10	-	hold

This genotyping protocol is used for the following strains:

Stock Number	Strain Name
002616	B6.129S7-Hba^tm1Paz/J
003295	B6;129-Ednrb^tm1Ywa/J
003312	B6.129S7-Ngf^tm1Gne/J
004861	B6EiC3Sn-Ts(16C-tel)1Cje/DnJ
005032	B6 x B6Ei.129(Cg)-Bmp8b^tm1Blh/EiJ
005429	B6.129S-Gpam^tm1Rcol/J
005670	B6.Cg-Gt(ROSA)26Sor^{tm1(rtTA,EGFP)Nagy}/J
005814	B6.129P2-Grm1^tm1Dgen/J
005819	B6.129-Insl5^tm1Dgen/J
005865	STOCK Tg(YACW408A5)1952Ricc/J
006833	B6.129-Sgcb^tm1Kcam/2J
006904	B6;129-Msc^tm1Eno/J
006955	B6.129S4(FVB)-Insr^tm1Khn/J
007005	129S-Scg5^tm1Led/J
008312	CBy.129P2(B6)-Tcrd^tm1Mom/SzJ
009662	B6.129X1-Ppargc1a^tm1Dpk/J
009685	B6N.129S1(Cg)-Tnks^tm1.1Yjc/J
010671	B6.129S4-Pkd1^tm2Ggg/J
010673	B6;129-Runx1^tm3.1Spe/J
010948	B6;129-Gabra1^tm4Geh/J
011081	B6;129-Osmr^tm1.1Nat/J
011082	B6;129-Edn2^tm1.1Nat/J
012560	129-Zic4^tm2Kjmi/J
012623	B6;129S4-Carf^tm1.1Aew/J
012653	B6;129-Pax7^tm1.1Fan/J
012839	B6.129X1(Cg)-Tnfrsf4^tm2(cre)Nik/J
012942	B6.129-Gabra2^tm1.1Geh/J
013717	B6.Cg-Slc20a1^tm1.2Cmg/J
014593	C;129S4-Rag2^tm1.1Flv Il2rg^tm1.1Flv/J
014594	C;129S4-Rag2^tm1.1Flv Thpo^{tm1.1(TPO)Flv} Il2rg^tm1.1Flv/J
014595	C;129S4-Rag2^tm1.1Flv Csf2/Il3^{tm1.1(CSF2,IL3)Flv} Il2rg^tm1.1Flv/J
017977	B6.129-Ulk2^tm1Thsn/J

Genotyping resources and troubleshooting
This page contains guidelines, frequently asked questions, and helpful external links to genotyping and PCR resources.