Haynes Lab:Notebook/Engineering PC-TFs/2013/03/05: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Engineering PC-TFs</span>
|style="background-color: #FFCC00"|<br>[[Image:Hayneslab3.gif|200px|center]][[Image:Asu logo 3.gif|200px|center]]<span style="font-size:22px;"><br></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: #800000" 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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* BL09
* BL09


''B)Follow digest procedure:''
<font size=3>''B)Follow digest procedure:''


   DNA          20.0 µL
   DNA          20.0 µL
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   dH2O        5.0  uL
   dH2O        5.0  uL
              
              
               = 30.0 uL
               = 30.0 uL </font>




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[[Image:gel3_5_2013.jpg|300px|right|Stock digest 3/5/2013]]
[[Image:gel3_5_2013.jpg|200px|Stock digest 3/5/2013]]
[[Image:gel3_5_2013_cutout.jpg|300px|left|Stock digest 3/5/2013]]
[[Image:gel3_5_2013_cutout.jpg|200px|Stock digest 3/5/2013]]
<br>
<br>
*<font size=4>''Transformation of hPCD, fshPCD, flyPCD''
<font size=4>''Transformation of hPCD, fshPCD, flyPCD''</font>
 
<br>
*Warm 100 μg/mL Amp agar plates at 37 °C
*Thaw fresh tube of DH5α Turbo cells on ice
*Add 30 μL of DH5α Turbo cells to DNA.
*Incubate cells + DNA on ice for 5 min.
*Label pre-warmed plates
*Transfer cells + DNA onto agar
*Add 10 - 15 sterile glass beads, shake, discard beads
*Incubate plates at 37 °C overnight


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Latest revision as of 22:31, 26 September 2017



 		Main project page
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Summary

  • Procedure

A) Get biobricks and enzymes from freezer.

SpeI, PstI, XbaI

The following list of parts are:


  • BL01
  • BL05
  • BL09

B)Follow digest procedure: 

  DNA          20.0 µL
  Enzyme 1     1.0  µL
  Enzyme 2     1.0  µL
  10x buffer   3.0  µL
  dH2O         5.0  uL
            
             = 30.0 uL


Digest Order:

Biobrick Restriction Enzymes

  • 1. BL01 XbaI/PstI
  • 2. BL05 XbaI/PstI
  • 3. BL09 XbaI/PstI


Stock digest 3/5/2013 Stock digest 3/5/2013
Transformation of hPCD, fshPCD, flyPCD

  • Warm 100 μg/mL Amp agar plates at 37 °C
  • Thaw fresh tube of DH5α Turbo cells on ice
  • Add 30 μL of DH5α Turbo cells to DNA.
  • Incubate cells + DNA on ice for 5 min.
  • Label pre-warmed plates
  • Transfer cells + DNA onto agar
  • Add 10 - 15 sterile glass beads, shake, discard beads
  • Incubate plates at 37 °C overnight


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