From 2007.igem.org
yesterday -- tomorrow
Digestion Reaction
PCR reaction
| PCR : PrefixBB-frt1-CmR-frt2-SufixBB
|
|
|
|
|
|
|
|
|
| PCR Settings
|
| Buffer (5x)
| (5x) 10µL
|
| Expected size
|
| Annealing (°C)
|
| MgCl2 10µM
| 10µM 0µL (no extra)
|
| 1000bp
|
| /
|
| dNTP 10µM
| 10µM 1µL
|
| Success
|
| Time Elongation
|
| Oligo F 10µM
| O54
| 10µM 4µL
|
| YES
|
| 72°C 1'40"
|
| Oligo R 10µM
| O55
| 10µM 4µL
|
| Image (click to enlarge)
|
| Number cycles
|
| Water
| qsp 50µL
|
| [[Image:|30px]]
|
| 30
|
| Polymerase
| Phusion 0,5µL
|
| Band (0=ladder)
|
|
|
| DNA
| pY-2P-IntC 5µL
|
|
|
| PCR : PrefixBB-frt1-CmR-frt2-SufixBB
|
|
|
|
|
|
|
|
|
| PCR Settings
|
| Buffer (5x)
| (5x) 10µL ! GC one !
|
| Expected size
|
| Annealing (°C)
|
| MgCl2 10µM
| 10µM 0µL (no extra)
|
| 1000bp
|
| /
|
| dNTP 10µM
| 10µM 1µL
|
| Success
|
| Time Elongation
|
| Oligo F 10µM
| O54
| 10µM 4µL
|
| NO
|
| 72°C 1'40"
|
| Oligo R 10µM
| O55
| 10µM 4µL
|
| Image (click to enlarge)
|
| Number cycles
|
| Water
| qsp 50µL
|
| [[Image:|30px]]
|
| 30
|
| Polymerase
| Phusion 0,5µL
|
| Band (0=ladder)
|
|
|
| DNA
| pY-2P-IntC 5µL
|
|
|
| PCR : PrefixBB-frt1-CmR-frt2-SufixBB
|
|
|
|
|
|
|
|
|
| PCR Settings
|
| Buffer (5x)
| (5x) 10µL ! GC one !
|
| Expected size
|
| Annealing (°C)
|
| MgCl2 10µM
| 10µM 0µL (no extra)
|
| 1000bp
|
| /
|
| dNTP 10µM
| 10µM 1µL
|
| Success
|
| Time Elongation
|
| Oligo F 10µM
| O54
| 10µM 4µL
|
| NO
|
| 72°C 1'40"
|
| Oligo R 10µM
| O55
| 10µM 4µL
|
| Image (click to enlarge)
|
| Number cycles
|
| Water
| qsp 50µL
|
| [[Image:|30px]]
|
| 30
|
| Polymerase
| Phusion 0,5µL
|
| Band (0=ladder)
|
|
|
| DNA
| pY-2P-IntC 5µL
|
|
|
| PCR : PrefixBB-frt1-CmR-frt2-T1T2-ufixBB
|
|
|
|
|
|
|
|
|
| PCR Settings
|
| Buffer (5x)
| (5x) 10µL
|
| Expected size
|
| Annealing (°C)
|
| MgCl2 10µM
| 10µM 0µL (no extra)
|
| 1000bp
|
| /
|
| dNTP 10µM
| 10µM 1µL
|
| Success
|
| Time Elongation
|
| Oligo F 10µM
| O54
| 10µM 4µL
|
| YES
|
| 72°C 1'40"
|
| Oligo R 10µM
| O56
| 10µM 4µL
|
| Image (click to enlarge)
|
| Number cycles
|
| Water
| qsp 50µL
|
| [[Image:|30px]]
|
| 30
|
| Polymerase
| Phusion 0,5µL
|
| Band (0=ladder)
|
|
|
| DNA
| pY-2P-IntC 5µL
|
|
|
| PCR : PrefixBB-frt1-CmR-frt2-T1T2-SufixBB
|
|
|
|
|
|
|
|
|
| PCR Settings
|
| Buffer (5x)
| (5x) 10µL ! GC one !
|
| Expected size
|
| Annealing (°C)
|
| MgCl2 10µM
| 10µM 0µL (no extra)
|
| 1000bp
|
| /
|
| dNTP 10µM
| 10µM 1µL
|
| Success
|
| Time Elongation
|
| Oligo F 10µM
| O54
| 10µM 4µL
|
| NO
|
| 72°C 1'40"
|
| Oligo R 10µM
| O56
| 10µM 4µL
|
| Image (click to enlarge)
|
| Number cycles
|
| Water
| qsp 50µL
|
| [[Image:|30px]]
|
| 30
|
| Polymerase
| Phusion 0,5µL
|
| Band (0=ladder)
|
|
|
| DNA
| pY-2P-IntC 5µL
|
|
|
| PCR : PrefixBB-frt1-CmR-frt2-T1T2-SufixBB
|
|
|
|
|
|
|
|
|
| PCR Settings
|
| Buffer (5x)
| (5x) 10µL ! GC one !
|
| Expected size
|
| Annealing (°C)
|
| MgCl2 10µM
| 10µM 0µL (no extra)
|
| 1000bp
|
| /
|
| dNTP 10µM
| 10µM 1µL
|
| Success
|
| Time Elongation
|
| Oligo F 10µM
| O54
| 10µM 4µL
|
| YES
|
| 72°C 1'40"
|
| Oligo R 10µM
| O56
| 10µM 4µL
|
| Image (click to enlarge)
|
| Number cycles
|
| Water
| qsp 50µL
|
| [[Image:|30px]]
|
| 30
|
| Polymerase
| Phusion 0,5µL
|
| Band (0=ladder)
|
|
|
| DNA
| pY-2P-IntC 5µL
|
|
|
Purification of Digestion and PCR products
Ligation Reactions
Culture
Launching of 80mL culture of the strain countaining the plasmid pY-2P-IntC, last minipreps revealed the weak nomber of plasmid recover with a classic miniprep on 5mL culture.
