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

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:igem-logo-150px.png|150px]]<span style="font-size:22px;"> iGEM Project name 1</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==Entry title==
=='''Friday 25th June 2010'''==
* Insert content here...
I did again the 3 ligations:<br />
1) With more GFP (small part) approximately 3:1 ratio:<br />
GFP (X/P)....................5ul<br />
cI plasmid (S/P)..........2ul<br />
H2O.........................10ul<br />
T4 ligase Buffer.........2ul<br />
T4 ligase....................1ul<br />
Total........................20ul<br />
<br />
2) With approximately equimolar concentrations of the parts:<br />
GFP (X/P)..................3ul<br />
cI plasmid (S/P)........4ul<br />
H2O........................10ul<br />
T4 ligase Buffer........2ul<br />
T4 ligase..................1ul<br />
Total.......................20ul<br />
<br />
 
*Ligation Plasmid 18 + cI-PCR + GFP<br />
3) With more or less equimolar concentrations <br />
 
cI-PCR (E/S).................2ul<br />
GFP(X/P)......................4ul<br />
Plasmid 18 (E/P) 3ul<br />
H2O 8ul<br />
T4 ligase Buffer 2ul<br />
T4 ligase 1ul<br />
Total 20ul<br />
<br />
 
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.<br />
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.<br />
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:<br />
*1 and 2 with Kanamycin <br />
*3 with Tetracycline<br />
*4 (GFP) with Ampicillin <br />
Those were left in the incubator at 37ºC overnight.<br />
<br />
 




Revision as of 18:04, 29 June 2010

iGEM Project name 1 <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

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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