User:Jorge E. Buendia Buendia/Notebook/iGEM UNAM-Genomics-Mexico/2010/07/02

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July 2nd, 2010
1. Make XbaI-PstI double restriction to GFP BBa_K145015 (74 min) extracted by PCR.
 * 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

2. Inactivate enzyme from XbaI-PstI double restriction to GFP E0240, made on July 1st. 80ºC for 20 min.

3. Dephosphate p30-MinBP PstI-SpeI, this plasmid will be ligated to GFP E0240 XbaI-PstI. Incubate 20 min at 37ºC and 10 min at 65ºC:
 * DNA -> 15ul
 * Buffer -> 3ul
 * Phophatase -> 1ul
 * HPLC -> 11ul
 * Total volume: 30ul

4. Inactivate enzyme from XbaI-PstI double restriction to GFP BBa_K145015 (74 min), 80ºC for 20 min.

5. Run gel to verify parts to be ligated:
 * 1) Dephosphated p30-MinBP SpeI-PstI
 * 2) GFP E0240 XbaI-PstI
 * 3) Dephosphated backbone pSB3K3 EcoRI-PstI
 * 4) Blue Promoter (extracted by PCR) EcoRI-SpeI
 * 5) GFP BBa_K145015 (74 min) XbaI-PstI
 * 6) Dephosphated p18 XbaI-PstI



GEL WAS A TOTAL FAIL, but I’ll even do ligations.

6. Make ligations:
 * 1. Dephosphated p30-MinBP SpeI-PstI (3ul) + GFP E0240 XbaI-PstI (6ul)
 * 4. Dephosphated backbone plasmid pSB3K3 EcoRI-PstI restriction (3ul) + Blue Promoter EcoRI-SpeI restriction (5ul) + GFP BBa_K145015 XbaI-PstI restriction (5ul)
 * 5. Dephosphated plasmid 18 XbaI-PstI restriction (3ul) + GFP BBa_K145015 XbaI-PstI restriction (5ul)


 * 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.

7. Replate transformation Backbone pSB3K3 + Blue Promoter + GFP E0240, this transformation was done on June 29th.


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