User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/06/16

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June 16th, 2010

1. Run gel to verify PCR from june 15th, it should’ve amplified the whole plasmid and contain the minimum blue promoter.

Lanes: 1,6) Ladder; 2) Minimum Blue Promoter 1 (MBP-1); 3) MBP-2; 4,5) Augusto’s samples.


2. Replate strain DH5-α in solid and liquid medium, we have to make competent cells with this strain, it has a different membrane and better transformation efficiency. This cells over-grew.


3. Repeat PCR with primers to amplify plasmid 30 and insert blue promoter into this plasmid.

  • PCR was done with plasmid 30 extracted by me (p30) and the one extracted by Mariana (p30M), I also used BBa_K137019 (2855 bp) as positive control for primers (C+) and water as negative control (C-).
  • I made PCR with Taq Polymerase, Platinum Taq and rTth Polymerase to test the enzymes.
  • Reactives needed for one reaction are as follows (I prepared 4 reactions, p30, p30M an negative control, positive control was done with preffix FWD and suffix REV primers).
  • PCR with Taq DNA polymerase
Reactive (ul)
Taq Polymerase -> 1
Taq Reaction Buffer -> 5
MgCl 50mM (can be used up to 3ul) -> 2.5
dNTP’s 0.4ug/ul -> 2.5
Primer Forward (can be used up to 3ul) -> 2.5
Primer Reverse (can be used up to 3ul) -> 2.5
HPLC -> 30
DNA -> 4
Total volume -> 50
  • PCR with RTTH polymerase
Reaction 1 (ul)
HPLC -> 10
Buffer 3.3x -> 6
Mg(Ac)2 -> 3
dNTP’s -> 5
Primer FWD -> 3
Primer REV -> 3
DNA -> 4
Total volume -> 30
Reaction 2 (ul)
HPLC -> 10.5
Buffer 3.3x -> 9
RTTH -> 0.5
Total -> 20
  • PCR with Platinum Taq DNA polymerase (ul)
10X PCR Buffer minus M -> 5
10mM dNTP mixture -> 1
50mM MgCl2 -> 1.5
Primer mix (10uM each) -> 1
Platinum Taq DNA Pol -> 0.2
Template DNA -> 4
HPLC -> 37.3
Total Volume -> 50


4. Re-plate plasmid 17, 18 and 30 to store as stock.



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