User:Jose Fabricio Lopez/Notebook/Logbook/2011/09/20: Difference between revisions

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==PCRs==
==PCRs==
*We are going to try a new approach for this PCRs. Now the time for First Denaturalization is the same [[User:Jose Fabricio Lopez/Notebook/Logbook/2011/09/1| to these PCRs]]
*We are going to try a new approach for this PCRs. Now the time for First Denaturalization is the same [[User:Jose Fabricio Lopez/Notebook/Logbook/2011/09/15| to these PCRs]]
*Now we use lower and higher annealing temperatures.
*Now we use lower and higher annealing temperatures.
 
*These amplifications have double terminador [[http://partsregistry.org/Part:BBa_B0015 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.


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‡Eluted from 1:100 from Vladimir´s [[User:Eduardo Vladimir Munoz/Notebook/UNAM Genomics Mexico 2011/2011/09/08#Gel| extraction stock 3]]
‡Eluted from 1:100 from Vladimir´s [[User:Eduardo Vladimir Munoz/Notebook/UNAM Genomics Mexico 2011/2011/09/08#Gel| extraction stock 3]]
†.5μL of Polymerase to fill 50μL in each reaction.
†.5μL of Polymerase to fill 50μL in each reaction.


Revision as of 09:55, 22 September 2011

iGEM 2011 <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Goals

  • PCR of Ter1-Backbone and Ter2-Backbone.
  • Band Extraction for DNA extracted in band (double extraction).

PCRs

  • 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.



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