From 2007.igem.org
yesterday -- tomorrow
Digestion Reaction
PCR reaction
| PCR : PrefixBB-frt1-CmR-frt2-SufixBB
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| 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
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| 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
| Ligations
|
| Number
| Insert
| Insert Volume (µL)
| Vector
| Vector Volume (µL)
| Comments
| Number of colonies
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| C22
|
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| D47
| 3
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| 0
|
| C23
|
|
| D72
| 3
|
| 0
|
| C24
|
|
| D72 (from Oct. 14)
| 3
|
| 0
|
| C25
|
|
| D50
| 3
|
| 0
|
| C26
|
|
| D41
| 3
|
| 0
|
| C27
|
|
| D22
| 5
|
| 0
|
| C28
|
|
| D81
| 11
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| 0
|
| C29
|
|
| D89
| 3
|
| 0
|
| C30
|
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| D90
| 3
|
| 0
|
| L68
| D56 - araC/pBad>>RBS-Cre BBinsert
| 10
| D72 - pSB2K3 BBvector
| 3
|
| 0
|
| L68 bis
| D56 (14/10/07) - araC/pBad>>RBS-Cre BBinsert
| 10
| D72 (14/10/07) - pSB2K3 BBvector
| 3
|
| 0
|
| L69
| D92
| 10
| D50
| 3
|
| 0
|
| L70
| D92
| 10
| D41
| 3
|
| 0
|
| L71
| D93
| 15
| D50
| 3
|
| 0
|
| L72
| D93
| 15
| D41
| 3
|
| 0
|
| L73
| D84
| 10
| D22
| 5
|
| 0
|
| L74
| D80
| 3
| D81
| 15
|
| 0
|
| L74 bis
| D80
| 4
| D81
| 12
|
| 0
|
| L75
| D83
| 4
| D89
| 3
|
| 0
|
| L76
| D83
| 10
| D90
| 3
|
| 0
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| L77
| D94.6
| 3
| D95
| 10
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| 0
|
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.
