Difference between revisions of "IGEM:Arizona State/2014/Notebook/asu igem 2014/2014/08/04"

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(Preparing another RBS-TesA and LacI Ligation)
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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:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</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"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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Latest revision as of 23:09, 26 September 2017

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Preparing another RBS-TesA and LacI Ligation

  • Digested more of RBS-TesA with X and P(insert). Digested more of LacI with S and P (backbone).
TesA+RBS insert with LacI+backbone
Reagent RBS-TesA (μL) LacI (μL)
DNA 15.0 15.0
10X fast Digest Buffer 2.0 2.0
PstI 1.0 1.0
XbaI 1.0 .0
SpeI 0.0 1.0
H2O 1.0 1.0
37°C for 1 hour
  • Both samples used in the Clean and Concentrate kit
  • Treat the LacI with Antarctic Phsophotase.
Antarctic Phsophotase Treatment
LacI DNA 10.0
Antarctic Phosphotase 1.0
Phosphotase Buffer 2.0
H2O 7.0
37°C for 1 hour then 65°C for 5 min
  • The RBS-TesA and LacI DNA samples were quantified:
  • RBS-TesA: 39.157 ng/μL
  • LacI: 62.579 ng/μL
  • Overnight ligation reactions were conducted for the test ligation and the negative control.
TesA+RBS insert with LacI+backbone
Reagent 1 (μL) Control (μL)
TesA+RBS 1.25 0.0
LacI 1.0 1.0
Ligation Buffer 1.0 1.0
Ligase 1.0 1.0
H2O 5.75 7.0
16°C overnight


  • Started seeds of 3 seperate colonies of ethanol plasmid
    • 50 ml LB w/ Chloramphenicol + picked colony
    • Cells didn't grow fast enough to add IPTG to start protein growth, so cells were left overnight to grow more