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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