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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