Mimulus:Microsatellite recipes


 * The original reference for the AAT markers: Kelly and Willis 1998
 * Check out the Mimulus Biological Resource Center for map location of these microsatellies.
 * The primers for these microsatellites are archived in genbank.
 * See the Full Microsatellite Sequences.
 * In addition to these recipes, check out the PCR Program for Microsatellites.
 * Contact David Lowry (dbl2@duke.edu) or other members of the Mimulus Community for more inforamtion about these markers.
 * [[Media:Microsatellite_PCR_conditions.xls|Microsat conditions spreadsheet]]

Below are the recipes that have been fine-tuned for Mimulus guttatus

AAT 9
Dye: VIC

Annealing Temp: 55

Cycles: 26


 * H20 8.05μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)	1.2μl
 * dNTPs (2.5mM)	1.2μl
 * AAT9R			0.6μl
 * AAT9F			0.25μl
 * Taq			0.2μl

AAT 39
Dye: FAM

Annealing Temp		53

Cycles			25


 * H20			5.05μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		1.2μl
 * dNTPs (.25mM)		1.2μl
 * 39R			0.6μl
 * 39F			0.25μl
 * Taq			0.2μl

AAT 211
Dye: FAM

Annealing Temp: 53

Cycles: 22
 * H20			5.05μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		1.2μl
 * dNTPs (.25mM)		1.2μl
 * 211R			0.6μl
 * 211F			0.25μl
 * Taq			0.2μl

AAT 217
Dye: FAM

Annealing Temp:		54

Cycles:			27


 * H20			8.05μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		1.2μl
 * dNTPs (.25mM)		1.2μl
 * 217R			0.6μl
 * 217F			0.25μl
 * Taq			0.2μl

AAT 222
Dye: NED

Annealing Temp		53

Cycles:			30
 * H20			5.05μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		1.2μl
 * dNTPs (.25mM)		1.2μl
 * 222R			0.6μl
 * 222F			0.25μl
 * Taq			0.2μl

AAT 225
Dye: VIC

Annealing Temp:		53

Cycles:			32


 * H20			8.05μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		1.2μl
 * dNTPs (.25mM)		1.2μl
 * 225R			0.6μl
 * 225F			0.25μl
 * Taq			0.2μl

AAT 230
Dye: VIC

Annealing Temp:		54

Cycles:			28


 * H20			6.85μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		2.4μl
 * dNTPs (.25mM)		1.2μl
 * 230R			0.6μl
 * 230F			0.25μl
 * Taq			0.2μl

AAT 233
Dye: FAM

Annealing Temp:		53

Cycles:			28


 * H20			5.05μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		1.2μl
 * dNTPs (.25mM)		1.2μl
 * 233R			0.6μl
 * 233F			0.25μl
 * Taq			0.2μl

AAT 240
Dye: FAM

Annealing Temp:		54

Cycles:			25


 * H20			7.45μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)	1.8μl
 * dNTPs (.25mM)	1.2μl
 * 240R			0.6μl
 * 240F			0.25μl
 * Taq			0.2μl

AAT 242
Dye: FAM

Annealing Temp:		49

Cycles:			32


 * H20			5.05μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		1.2μl
 * dNTPs (.25mM)		1.2μl
 * 242R			0.6μl
 * 242F			0.25μl
 * Taq			0.2μl

AAT 261
Dye: VIC

Annealing Temp:	49

Cycles:			30


 * H20			4.45μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		1.8μl
 * dNTPs (.25mM)		1.2μl
 * 261R			0.6μl
 * 261F			0.25μl
 * Taq			0.2μl

AAT 265
Dye: NED

Annealing Temp:		55

Cycles:		20


 * H20			5.05μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		1.2μl
 * dNTPs (.25mM)		1.2μl
 * 265R			0.6μl
 * 265F			0.25μl
 * Taq			0.2μl

AAT 267
Dye: VIC

Annealing Temp:		49

Cycles: 30


 * H20			6.85μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		2.4μl
 * dNTPs (.25mM)		1.2μl
 * 267R			0.6μl
 * 267F			0.25μl
 * Taq			0.2μl

AAT 272
Dye: NED

Annealing Temp:		54

Cycles:			30


 * H20			4.45μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		1.8μl
 * dNTPs (.25mM)		1.2μl
 * 272R			0.6μl
 * 272F			0.25μl
 * Taq			0.2μl

AAT 278
Dye: NED

Annealing Temp:		53

Cycles:			24


 * H20			5.95μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		1.8μl
 * KCl (0.5M)		1.5μl
 * dNTPs (.25mM)		1.2μl
 * 278R			0.6μl
 * 278F			0.25μl
 * Taq			0.2μl

AAT 283
Dye: VIC

Annealing Temp:		52

Cycles:			29


 * H20			3.85μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		2.4μl
 * dNTPs (.25mM)		1.2μl
 * 283R			0.6μl
 * 283F			0.25μl
 * Taq			0.2μl

AAT 296
Dye: NED

Annealing Temp:		53

Cycles:			28


 * H20			2.1μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		1.8μl
 * KCl (.5M)			1.5μl
 * dNTPs (.25mM)		1.2μl
 * 296R		1.2μl
 * 296F		0.5μl
 * Taq			0.2μl

AAT 300
Dye: FAM

Annealing Temp		55

Cycles			22


 * H20			8.05μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		1.2μl
 * dNTPs (.25mM)		1.2μl
 * 300R			0.6μl
 * 300F			0.25μl
 * Taq			0.2μl

AAT 308
Dye: VIC

Annealing Temp:		55

Cycles:			24


 * H20			5.05μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		1.2μl
 * dNTPs (.25mM)		1.2μl
 * 308R			0.6μl
 * 308F			0.25μl
 * Taq			0.2μl

AAT 312
Dye: NED

Annealing Temp:		51

Cycles:			30
 * H20			2.95μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		1.8μl
 * KCl (0.5M)		1.5μl
 * dNTPs (.25mM)		1.2μl
 * 312R			0.6μl
 * 312F			0.25μl
 * Taq			0.2μl

AAT 333
Dye: VIC

Annealing Temp:		51

Cycles:			30


 * H20			3.85μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		2.4μl
 * dNTPs (.25mM)		1.2μl
 * 333R			0.6μl
 * Taq			0.2μl

AAT 350
Dye: FAM

Annealing Temp:		52

Cycles:			28


 * H20			5.05μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		1.2μl
 * dNTPs (.25mM)		1.2μl
 * 350R			0.6μl
 * 350F			0.25μl
 * Taq			0.2μl

AAT 356
Dye: VIC

Annealing Temp:		55

Cycles:			25
 * H20			8.05μl
 * 10X Buffer		1.5μl
 * MgCl2 (25mM)		1.2μl
 * dNTPs (.25mM)		1.2μl
 * 356R			0.6μl
 * 356F			0.25μl
 * Taq			0.2μl

AAT 364- NED

•	Annealing Temp		53 •	Cycles			30

per sample	x15		x110 •	H20			3.7μl		55.5μl		407μl •	10X Buffer		1.5μl		22.5μl		165μl •	MgCl2 (25mM)		1.8μl		27μl		198μl •	KCl (.5M)			0.75μl		11.25μl	82.5μl •	dNTPs (.25mM)		1.2μl 		18μl		132μl •	364R			0.6μl		9μl		66μl •	364F			0.25μl		3.75μl		27.5μl •	Taq			0.2μl		3μl		22μl

AAT 367 - VIC

•	Annealing Temp		54 •	Cycles			22

per sample	x15		x110 •	H20			4.45μl		66.75μl	489.5μl •	10X Buffer		1.5μl		22.5μl		165μl •	MgCl2 (25mM)		1.8μl		27μl		198μl •	dNTPs (.25mM)		1.2μl 		18μl		132μl •	367R			0.6μl		9μl		66μl •	367F			0.25μl		3.75μl		27.5μl •	Taq			0.2μl		3μl		22μl

AAT 372- NED

•	Annealing Temp		55 •	Cycles			25

per sample	x15		x110 •	H20			4.45μl		66.75μl	621.5μl •	10X Buffer		1.5μl		22.5μl		165μl •	MgCl2 (25mM)		0.6μl		9μl		66μl •	dNTPs (.25mM)		1.2μl 		18μl		132μl •	372R			0.6μl		9μl		66μl •	372F			0.25μl		3.75μl		27.5μl •	Taq			0.2μl		3μl		22μl

AAT 374 - FAM

•	Annealing Temp		53 •	Cycles			28

per sample	x15		x110 •	H20			4.45μl		66.75μl	489.5μl •	10X Buffer		1.5μl		22.5μl		165μl •	MgCl2 (25mM)		1.8μl		27μl		198μl •	dNTPs (.25mM)		1.2μl 		18μl		132μl •	374R			0.6μl		9μl		66μl •	374F			0.25μl		3.75μl		27.5μl •	Taq			0.2μl		3μl		22μl

AG 19- NED

•	Annealing Temp		53 •	Cycles			30

per sample	x15		x110 •	H20			5.65μl		84.75μl	621.5μl •	10X Buffer		1.5μl		22.5μl		165μl •	MgCl2 (25mM)		0.6μl		9μl		66μl •	dNTPs (.25mM)		1.2μl 		18μl		132μl •	AG19R			0.6μl		9μl		66μl •	AG19F			0.25μl		3.75μl		27.5μl •	Taq			0.2μl		3μl		22μl

ap3-VIC

•	Annealing Temp		53 •	Cycles			25

per sample	x15		x110 •	H20			5.1μl		76.5μl		561μl •	10X Buffer		1.5μl		22.5μl		165μl •	MgCl2 (25mM)		1.2μl		18μl		132μl •	dNTPs (.25mM)		1.2μl 		18μl		132μl •	AP3R			0.6μl		9μl		66μl •	AP3F			0.25μl		3.75μl		27.5μl •	Taq			0.2μl		3μl		22μl

lfy- NED

•	Annealing Temp		52 •	Cycles			19

per sample	x15		x110 •	H20			5.05μl		75.75μl	555.5μl •	10X Buffer		1.5μl		22.5μl		165μl •	MgCl2 (25mM)		1.2μl		18μl		132μl •	dNTPs (.25mM)		1.2μl 		18μl		132μl •	LFYR			0.6μl		9μl		66μl •	LFYF			0.25μl		3.75μl		27.5μl •	Taq			0.2μl		3μl		22μl

CYCB - FAM

•	Annealing temp		62 •	cycles			16

per sample	x15		x110 •	H20			5.05μl		75.75μl	555.5μl •	10X Buffer		1.5μl		22.5μl		165μl •	MgCl2 (25mM)		1.2μl		18μl		132μl •	dNTPs (.25mM)		1.2μl 		18μl		132μl •	CYCBR			0.6μl		9μl		66μl •	CYCBF			0.25μl		3.75μl		27.5μl •	Taq			0.2μl		3μl		22μl

CYCA - FAM

•	Annealing temp		58 •	cycles			37

per sample	x15		x110 •	H20			4.05μl		60.75μl	445.5μl •	10X Buffer		1.5μl		22.5μl		165μl •	MgCl2 (25mM)		1.8μl		27μl		198μl •	dNTPs (.25mM)		1.5μl 		22.5μl		165μl •	CYCAR			0.65μl		9.75μl		71.5μl •	CYCAF			0.3μl		4.5μl		33μl •	Taq			0.2μl		3μl		22μl