User:Jose Fabricio Lopez/Notebook/Logbook/2011/09/20

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iGEM 2011 Main project page
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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 Mix 1 (DMSO) Ter1 Mix 2 (DMSO) Ter2 Mix 3 (NoDMSO)Ter1 Mix 4 (NoDMSO)Ter2
H2O 50 50 50 50
5x Phusion HF Buffer 20.2 20.2 20.2 20.2
dATP 2.1 2.1 2.1 2.1
dCTP 2.1 2.1 2.1 2.1
dTTP 2.1 2.1 2.1 2.1
dGTP 2.1 2.1 2.1 2.1
Primer fwd 10.1 10.1 10.1 10.1
Primer rev 10.1 10.1 10.1 10.1
DNA‡ 2.1 2.1 2.1 2.1
DMSO 3.0 3.0 NA NA
Phusion Pol† NA NA NA NA

‡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 Gel instruct. Gel

Initial

  • 95°C 5 min

30 Rep

  • 98°C 10s
  • 54.7°C & 60.3°C 30s
  • 72°C 2:00min

Hold

  • 72°C 7:00 min
  • 4°C ∞
  • 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)

‡Today´s Band extraction.

Band extraction

Quigen´s kit, eluted in 40μL. See picture above



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