Mimulus:Microsatellite recipes: Difference between revisions

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*In addition to these recipes, check out the [[PCR Program for Microsatellites]].   
*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.
*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'':
Below are the recipes that have been fine-tuned for ''Mimulus guttatus''


==AAT 9==
==AAT 9==
Line 15: Line 16:
Cycles: 26
Cycles: 26


*H20 5.05μl
*H20 8.05μl
*10X Buffer 1.5μl
*10X Buffer 1.5μl
*MgCl2 (25mM) 1.2μl
*MgCl2 (25mM) 1.2μl
Line 21: Line 22:
*AAT9R 0.6μl
*AAT9R 0.6μl
*AAT9F 0.25μl
*AAT9F 0.25μl
*Taq 0.2μl
*Taq 0.2μl
 


==AAT 39==
==AAT 39==
Line 63: Line 63:




*H20 5.05μl
*H20 8.05μl
*10X Buffer 1.5μl
*10X Buffer 1.5μl
*MgCl2 (25mM) 1.2μl
*MgCl2 (25mM) 1.2μl
Line 69: Line 69:
*217R 0.6μl
*217R 0.6μl
*217F 0.25μl
*217F 0.25μl
*Taq 0.2μl
*Taq 0.2μl
 


==AAT 222==
==AAT 222==
Line 93: Line 92:
Cycles: 32
Cycles: 32


*H20 5.05μl
*H20 8.05μl
*10X Buffer 1.5μl
*10X Buffer 1.5μl
*MgCl2 (25mM) 1.2μl
*MgCl2 (25mM) 1.2μl
Line 99: Line 98:
*225R 0.6μl
*225R 0.6μl
*225F 0.25μl
*225F 0.25μl
*Taq 0.2μl
*Taq 0.2μl
 
 


==AAT 230==  
==AAT 230==  
Line 111: Line 108:


*H20 3.85μl
*H20 6.85μl
*10X Buffer 1.5μl
*10X Buffer 1.5μl
*MgCl2 (25mM) 2.4μl
*MgCl2 (25mM) 2.4μl
Line 117: Line 114:
*230R 0.6μl
*230R 0.6μl
*230F 0.25μl
*230F 0.25μl
*Taq 0.2μl
*Taq 0.2μl
 


==AAT 233==  
==AAT 233==  
Line 142: Line 138:
Cycles: 25
Cycles: 25


*H20 4.45μl
*H20 7.45μl
*10X Buffer 1.5μl
*10X Buffer 1.5μl
*MgCl2 (25mM) 1.8μl
*MgCl2 (25mM) 1.8μl
Line 148: Line 144:
*240R 0.6μl
*240R 0.6μl
*240F 0.25μl
*240F 0.25μl
*Taq 0.2μl
*Taq 0.2μl
 


==AAT 242==
==AAT 242==
Line 207: Line 202:




Annealing Temp 49
Annealing Temp: 49


*H20 3.85μl
Cycles: 30
 
*H20 6.85μl
*10X Buffer 1.5μl
*10X Buffer 1.5μl
*MgCl2 (25mM) 2.4μl
*MgCl2 (25mM) 2.4μl
Line 215: Line 212:
*267R 0.6μl
*267R 0.6μl
*267F 0.25μl
*267F 0.25μl
*Taq 0.2μl
*Taq 0.2μl
 


==AAT 272==
==AAT 272==
Line 239: Line 235:


Annealing Temp: 53
Annealing Temp: 53
Cycles: 24
Cycles: 24


Line 244: Line 241:


*H20 2.95μl
*H20 5.95μl
*10X Buffer 1.5μl
*10X Buffer 1.5μl
*MgCl2 (25mM) 1.8μl
*MgCl2 (25mM) 1.8μl
Line 251: Line 248:
*278R 0.6μl
*278R 0.6μl
*278F 0.25μl
*278F 0.25μl
*Taq 0.2μl
*Taq 0.2μl
 


==AAT 283==
==AAT 283==
Line 273: Line 269:
Dye: NED
Dye: NED


Annealing Temp: 53


Cycles: 28


• 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




    per sample x15 x110
==AAT 300==
• H20 2.1μl 31.5μl 231μl
Dye: FAM
• 10X Buffer 1.5μl 22.5μl 165μl
• MgCl2 (25mM) 1.8μl 27μl 198μl
• KCl (.5M) 1.5μl 22.5μl 165μl
• dNTPs (.25mM) 1.2μl 18μl 132μl
• 296R ** 1.2μl 18μl 132μl **
• 296F ** 0.5μl 7.5μl 55μl **
• Taq 0.2μl 3μl 22μl


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 300 - FAM
==AAT 350==
Dye: FAM


Annealing Temp: 52


Cycles: 28


• Annealing Temp 55
*H20 5.05μl
• Cycles 22
*10X Buffer 1.5μl
*MgCl2 (25mM) 1.2μl
*dNTPs (.25mM) 1.2μl
*350R 0.6μl
*350F 0.25μl
*Taq 0.2μl






    per sample x15 x110
==AAT 356==
• H20 5.05μl 75.75μl 555.5μl
Dye: VIC
• 10X Buffer 1.5μl 22.5μl 165μl
• MgCl2 (25mM) 1.2μl 18μl 132μl
• dNTPs (.25mM) 1.2μl 18μl 132μl
• 300R 0.6μl 9μl 66μl
• 300F 0.25μl 3.75μl 27.5μl
• Taq 0.2μl 3μl 22μl
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
AAT 308 - VIC
 
 
 
• Annealing Temp 55
• Cycles 24
 
 
 
    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
• 308R 0.6μl 9μl 66μl
• 308F 0.25μl 3.75μl 27.5μl
• Taq 0.2μl 3μl 22μl
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
AAT 312 - NED
 
 
 
• Annealing Temp 51
• Cycles 30
 
 
    per sample x15 x110
• H20 2.95μl 44.25μl 324.5μl
• 10X Buffer 1.5μl 22.5μl 165μl
• MgCl2 (25mM) 1.8μl 27μl 198μl
• KCl (0.5M) 1.5μl 22.5μl 165μl
• dNTPs (.25mM) 1.2μl 18μl 132μl
• 312R 0.6μl 9μl 66μl
• 312F 0.25μl 3.75μl 27.5μl
• Taq 0.2μl 3μl 22μl
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
AAT 333 - VIC
 
 
 
• Annealing Temp 51
• Cycles 30
 
 
 
    per sample x15 x110
• H20 3.85μl 57.75μl 423.5μl
• 10X Buffer 1.5μl 22.5μl 165μl
• MgCl2 (25mM) 2.4μl 36μl 264μl
• dNTPs (.25mM) 1.2μl 18μl 132μl
• 333R 0.6μl 9μl 66μl
• 333F 0.25μl 3.75μl 27.5μl
• Taq 0.2μl 3μl 22μl
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
AAT 350 - FAM
 
 
 
• Annealing Temp 52
• Cycles 28
 
 
    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
• 350R 0.6μl 9μl 66μl
• 350F 0.25μl 3.75μl 27.5μl
• Taq 0.2μl 3μl 22μl
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
AAT 356- VIC
 
 
• Annealing Temp 55
• Cycles 25
 
 
 
 
    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
• 356R 0.6μl 9μl 66μl
• 356F 0.25μl 3.75μl 27.5μl
• Taq 0.2μl 3μl 22μl
 
 


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




Latest revision as of 21:37, 12 December 2006

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

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