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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