User:Jorge Arturo Zepeda/Notebook/iGem LCG-UNAM team 2010/2010/06/19

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Saturday 19th June 2010

I performed two ligations: cI inverter plasmid to GFP(with RBS) and Plasmid 18 to cI inverter PCR and GFP (with RBS), for this fist I had to check in an agarose gel the quantity of the DNA of each part, there are two approaches to ligate, one is to put equimolar quantities of all the parts, and the other is to put the small and the big parts in a ratio of 3:1.

The gel came out as follows:

1) Ladder
2) GFP
3) plasmid 18
4) cI inverter PCR
5) cI inverter plasmid

  • I did the ligation for the cI plasmid + GFP twice:


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 10 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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