Mimulus:Microsatellite recipes: Difference between revisions
No edit summary |
|||
| (15 intermediate revisions by 2 users not shown) | |||
| Line 5: | Line 5: | ||
*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 | *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 | *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 | *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 | *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 | *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 | ||
Cycles: 30 | |||
*H20 | *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 | *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 296: | Line 292: | ||
*H20 | *H20 8.05μl | ||
*10X Buffer 1.5μl | *10X Buffer 1.5μl | ||
*MgCl2 (25mM) 1.2μl | *MgCl2 (25mM) 1.2μl | ||
| Line 302: | Line 298: | ||
*300R 0.6μl | *300R 0.6μl | ||
*300F 0.25μl | *300F 0.25μl | ||
*Taq 0.2μl | *Taq 0.2μl | ||
==AAT 308== | ==AAT 308== | ||
| Line 339: | Line 334: | ||
==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 | |||
Latest revision as of 21:37, 12 December 2006
- 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.
- 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
