User:Jorge Arturo Zepeda/Notebook/iGem LCG-UNAM team 2010/2010/06/25

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Friday 25th June 2010

I did again the 3 ligations:
1) With more GFP (small part) approximately 3:1 ratio:
GFP (X/P)....................5ul
cI plasmid (S/P)..........2ul
H2O.........................10ul
T4 ligase Buffer.........2ul
T4 ligase....................1ul
Total........................20ul

2) With approximately equimolar concentrations of the parts:
GFP (X/P)..................3ul
cI plasmid (S/P)........4ul
H2O........................10ul
T4 ligase Buffer........2ul
T4 ligase..................1ul
Total.......................20ul

  • Ligation Plasmid 18 + cI-PCR + GFP

3) With more or less equimolar concentrations

cI-PCR (E/S).................2ul
GFP(X/P)......................4ul
Plasmid 18 (E/P) 3ul
H2O 8ul
T4 ligase Buffer 2ul
T4 ligase 1ul
Total 20ul

I left the ligations for 15 minutes at room temperature, then to inactivate the enzyme I gave them a heat shock at 62ºC for another 10 minutes.
After that I performed a heat shock transformation using 5 ul of the mix for each transformation, I also transformed a plasmid containing GFP as a positive control.
Then I grew them in 1ml of LB with no antibiotic for an hour for posterior plating in petri boxes with the respective antibiotic as follows:

  • 1 and 2 with Kanamycin
  • 3 with Tetracycline
  • 4 (GFP) with Ampicillin

Those were left in the incubator at 37ºC overnight.



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