User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/10/25: Difference between revisions

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==Entry title==
==October 25, 2010==
* Insert your content here.
1. Extract plasmid from colonies 1-10 incubated on October 24, the plasmid should contain: pSB3K3 + J23101 + ΔRBS + GFP E004
*Plamid extraction was made using the QIAprep Spin Miniprep Kit.


2. Made PCR to test pSB3K3 + J23101 + ΔRBS + GFP E004 construction.
*PCR will be done with Platinum Taq Polymerase.
*Primers used: Preffix FWD-Suffix REV.
*Template for the reaction is extracted plasmid from the transformation with this construction (Colonies 1-10, tubes are marked the same way).
*Positive control: BBa_I51020 (p19).
*'''PCR with Platinum Taq DNA polymerase -> Volume (ul)'''
:10X PCR Buffer minus M -> 5
:10mM dNTP mixture -> 1
:50mM MgCl2 -> 2.5
:Primer mix (10uM each) -> 2
:Platinum Taq DNA Pol -> 0.4
:Template DNA -> 1
:HPLC -> 38.1
:Total volume -> 50
*If primers are separated and in concentration 5uM, use 1ul of each one.
*'''Thermocycler program:'''
:1. 95ºC 5 min
:2. 35 cycles
::-95ºC 45 seg
::-55ºC 45 seg
::-72ºC 1:10 min
:3. 72ºC 10 min
:4. Hold 4ºC




Revision as of 15:37, 25 October 2010

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October 25, 2010

1. Extract plasmid from colonies 1-10 incubated on October 24, the plasmid should contain: pSB3K3 + J23101 + ΔRBS + GFP E004

  • Plamid extraction was made using the QIAprep Spin Miniprep Kit.


2. Made PCR to test pSB3K3 + J23101 + ΔRBS + GFP E004 construction.

  • PCR will be done with Platinum Taq Polymerase.
  • Primers used: Preffix FWD-Suffix REV.
  • Template for the reaction is extracted plasmid from the transformation with this construction (Colonies 1-10, tubes are marked the same way).
  • Positive control: BBa_I51020 (p19).
  • PCR with Platinum Taq DNA polymerase -> Volume (ul)
10X PCR Buffer minus M -> 5
10mM dNTP mixture -> 1
50mM MgCl2 -> 2.5
Primer mix (10uM each) -> 2
Platinum Taq DNA Pol -> 0.4
Template DNA -> 1
HPLC -> 38.1
Total volume -> 50
  • If primers are separated and in concentration 5uM, use 1ul of each one.
  • Thermocycler program:
1. 95ºC 5 min
2. 35 cycles
-95ºC 45 seg
-55ºC 45 seg
-72ºC 1:10 min
3. 72ºC 10 min
4. Hold 4ºC



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