User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/06/22

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July 22nd, 2010
1. Re-run gel to verify PCR from June 21th.



￼Lanes: 1) Ladder; 2) p30-minBP 1 rTth; 3) p30-minBP 2 rTth; 4) Ctrl 3 kb’s rTth; 5) p30-minBP 1 Platinum; 6) p30-minBP 2 Platinum; 7) Ctrl 3 kb’s Platinum.

2. Replate transformation with backbone plasmid pSB3K3-P1010 from June 21st in solid medium and incubate in liquid medium to extract plasmid, GFP reporter E0240 was also incubated in liquid medium to make plasmid extraction.

3. Make PCR to amplify plasmid 30 and ligate with Blue Promoter, PCR will be made with rTth and Taq Platinum. Reactives used are as follows:
 * PCR with RTTH polymerase
 * Reaction 1	(ul x sample)
 * HPLC -> 6
 * Buffer 3.3x -> 6
 * Mg(Ac)2 -> 3
 * dNTP’s -> 5
 * Primer FWD	-> 3
 * Primer REV -> 3
 * DNA -> 4
 * Total volume -> 30


 * Reaction 2
 * 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 -> 2.5
 * Primer mix (10uM each) -> 2
 * Platinum Taq DNA Pol -> 0.4
 * Template DNA -> 4
 * HPLC -> 35.1
 * Total volume -> 50


 * PCR tubes are marked 1-6:
 * 1: rTth11 p30-MinBP
 * 2: rTth Ctrl ~3 kb’s
 * 3: rTth Ctrl -
 * 4: Platinum Taq p30-minBP
 * 5: Platinum Taq Ctrl ~3 kb’s
 * 6: Platinum Taq Ctrl -

4. Extract plasmids Backbone pSB3K3-P1010 and GFP reporter E0240 using the High Pure Plasmid Isolation Kit Roche.


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