Goals
- PCR of Ter1-Backbone and Ter2-Backbone.
- Band Extraction for DNA extracted in band (double extraction).
Preparations
- We are going to try a new approach for this PCRs. Now the time for First Denaturalization is the same to these PCRs
- Now we use lower and higher annealing temperatures.
- These amplifications have double terminador [B0015] in pSB1AK3. We are going to get overlap with PFOR2 and HydG with Fwd primers (respective primers) and overlap with HydA and HydEF1 with reverse primers (respective primers), this includes prefix.
Reagents
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Mix 1 (DMSO) Ter1
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Mix 2 (DMSO) Ter2
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Mix 3 (NoDMSO)Ter1
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Mix 4 (NoDMSO)Ter2
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H2O
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50
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50
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50
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50
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5x Phusion HF Buffer
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20.2
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20.2
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20.2
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20.2
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dATP
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2.1
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2.1
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2.1
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2.1
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dCTP
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2.1
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2.1
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2.1
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2.1
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dTTP
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2.1
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2.1
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2.1
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2.1
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dGTP
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2.1
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2.1
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2.1
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2.1
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Primer fwd
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10.1
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10.1
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10.1
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10.1
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Primer rev
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10.1
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10.1
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10.1
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10.1
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DNA‡
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2.1
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2.1
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2.1
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2.1
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DMSO
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3.0
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3.0
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NA
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NA
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Phusion Pol†
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NA
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NA
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NA
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NA
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‡Eluted from 1:100 from Vladimir´s extraction stock 3
†.5μL of Polymerase to fill 50μL in each reaction.
- Each mix yields 2 reactions (1) 54.7°C and (2) 60.3°C for annealing.
PCR
Cycles
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Gel instruct.
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Gel
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Initial
30 Rep
- 98°C 10s
- 54.7°C & 60.3°C 30s
- 72°C 2:00min
Hold
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- Lane1 Ladder 500bp
- Lane2 PFOR2 E.B.‡
- Lane3 HydG E.B.‡
- Lane4 Ter1 DMSO (1)
- Lane5 Ter1 DMSO (2)
- Lane6 Empty
- Lane7 Ter2 DMSO (1)
- Lane8 Ter2 DMSO (2)
- Lane9 Ter1 Not DMSO (1)
- Lane10 Ter1 Not DMSO (2)
- Lane11 Ter2 Not DMSO (1)
- Lane12 Ter2 Not DMSO (2)
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‡Today´s Band extraction.
Band extraction
Quigen´s kit, eluted in 40μL.
See picture above
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