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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