User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/06/28

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June 28th, 2010
1. Run gel to verify restrictions from June 25th, p30-MinBP with SpeI and PstI-SpeI.



Lanes: 1) Ladder; 2) SpeI restriction p30-MinBP; 3) PstI-SpeI restriction p30-MinBP.

2. Dephosphate backbone plasmid, incubate 20 min at 37ºC and 10 min at 65ºC:
 * DNA -> 15ul
 * Buffer -> 3ul
 * Phophatase -> 1ul
 * HPLC -> 11ul
 * Total volume: 30ul

3. Restriction to plasmid 18 with XbaI-PstI.


 * Double restriction methods XbaI and PstI (DNA 10 ul, Total volume 30 ul):
 * DNA -> 10 ul
 * Buffer 3 -> 3 ul (10% of total volume)
 * BSA (required by SpeI) -> 1 ul
 * PstI -> 1.5 ul
 * XbaI -> 1.5 ul
 * HPLC -> 13 ul (to complete total volume of 30ul)
 * Incubate at 37ºC overnight

4. Dephosphate plasmid 18 to ligate with GFP BBa_K145015, incubate 20 min at 37ºC and 10 min at 65ºC:
 * DNA -> 15ul
 * Buffer -> 3ul
 * Phophatase -> 1ul
 * HPLC -> 11ul
 * Total volume: 30ul

5. Run gel to check parts that will be ligated.
 * 1) p30-MinBP SpeI restriction
 * 2) p30-MinBP SpeI-PstI restriction
 * 3) GFP E0240 XbaI-PstI restriction
 * 4) GFP BBa_K145015 XbaI-PstI restriction
 * 5) Dephosphated backbone plasmid pSB3K3 EcoRI-PstI restriction
 * 6) Blue Promoter EcoRI-SpeI restriction



6. Make ligations:
 * 1) p30-MinBP SpeI-PstI restrictionn (2ul) + GFP E0240 XbaI-PstI restriction (5ul)
 * 2) p30-MinBP SpeI-PstI restriction (2ul) + GFP BBa_K145015 XbaI-PstI restriction (5ul)
 * 3) Dephosphated backbone plasmid pSB3K3 EcoRI-PstI restriction (2ul) + Blue Promoter EcoRI-SpeI restriction (5ul) + GFP E0240 XbaI-PstI restriction (5ul)
 * 4) Dephosphated backbone plasmid pSB3K3 EcoRI-PstI restriction + Blue Promoter EcoRI-SpeI restriction + GFP BBa_K145015 XbaI-PstI restriction WE RAN OUT OF GFP E0240 XbaI-PstI #restriction SO I COULDN’T DO LIGATIONS 4 AND 5
 * 5) Dephosphated plasmid 18 XbaI-PstI restriction + GFP BBa_K145015 XbaI-PstI restriction
 * 6) Religate p30-MinBP SpeI restriction (2ul)


 * Ligation methods (Total volume 20ul):
 * DNA -> Depends on concentration
 * Buffer for T4 DNA ligase 10X -> 2ul (Final concentration 10%)
 * T4 DNA ligase -> 1ul
 * HPLC -> Complete total volume (20ul)
 * Incubate overnight at 16ºC
 * Mix well (vortex) buffer and reaction tubes.


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