Difference between revisions of "Haynes Lab:Notebook/Investigating Photo-Switchable Synthetic Nucleosomes/2013/04/25"

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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>}}
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|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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* Transform 10 μl of the Dpn I-treated DNA from each control and sample reaction into separate 50-μl aliquots of BL21 cells  
 
* Transform 10 μl of the Dpn I-treated DNA from each control and sample reaction into separate 50-μl aliquots of BL21 cells  
  
[[Image:VN_Mutagenesis_Transformation_Trial_1.png‎|thumb|frame|center|No colonies were seen the next day.]]
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[[Image:VN_Mutagenesis_Transformation_Trial_1_BL21.png‎|thumb|frame|center|No colonies were seen the next day.]]
  
 
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Latest revision as of 21:40, 26 September 2017

Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

April 25, 2013

  • QuickChange Site Directed Mutagenesis
  • Use 50 ng of dsDNA template and 125 ng of each primer.
  • Template strands are about 5kb for H2B-GFP and 7kb for LOV.
Reagents H2B LOV
Plasmid DNA (50 ng) 0.4 μL 0.2
primer 1 (10 μM, 125 ng) 1.7 1.7
primer 2 (10 μM, 125 ng) 1.7 1.7
10x reaction buffer 5.0 5.0
dNTP mix 1.0 1.0
dH2O 40.2 40.4
Total 50.0 50.0
  • Add 1 μl of PfuTurbo DNA polymerase (2.5 U/μl) to each control and sample reaction
  • Thermal cycling
    • 95°C/ 30 sec
    • [95°C/ 30 sec; 55°C/ 1 min; 68°C/ 7 min (1 min/kb plasmid length)]x18
    • 4°C, ∞
  • DpnI Digest (gets rid of methylated template DNA)
  1. H2B mut rxn
  2. H2B control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)
  3. LOV mut rxn
  4. LOV control (DNA, 10x buffer, Quick soln. in 25 μL; no PCR)
  • Add 1 μL DpnI enzyme to each sample
  • Gently and thoroughly mix each reaction, spin down in a microcentrifuge for 1 minute, and immediately incubate at 37°C for 1 hour to digest the parental supercoiled dsDNA
  • Transform 10 μl of the Dpn I-treated DNA from each control and sample reaction into separate 50-μl aliquots of BL21 cells
No colonies were seen the next day.