User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/19: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</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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| bgcolor=#cfcfcf | Volume  
| bgcolor=#cfcfcf | Volume  
| rowspan="7" | <u>Expected:</u><br>1,2. CMV-dCas9-HA = 5317, 4189<br>CMV-dCas9-VP64 =5459, 3016, 1337<br>
| rowspan="7" | <u>Expected:</u><br>1,2. CMV-dCas9-HA = 5317, 4189<br>CMV-dCas9-VP64 =5459, 3016, 1337<br>
| rowspan="7" | [[Image:KAH051915_gel1.jpg|250px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
| rowspan="7" | [[Image:KAH051915_gel1.jpg|150px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
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| DNA(plasmid) || 2.0 μL
| DNA(plasmid) || 2.0 μL
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--> 37°C/ ~10 min.
--> 37°C/ ~10 min.
CONCLUSIONS
* Success! Keep clone #1 (lane 1). Continue to next step: histone insertions




Latest revision as of 00:58, 27 September 2017

Karmella's BioBrick Cloning Main project page
Previous entry      Next entry

05/19/15

  • Cas-tone - cultures, minipreps, digests
  • Rene - PCR library 96-well plate


Cas-tone - cultures, minipreps, digests

  • Minipreps
    • Check with FD E/P digests
    • Modification (dsOligo insert replacing VP64) removes an EcoRI site
Reagent Volume Expected:
1,2. CMV-dCas9-HA = 5317, 4189
CMV-dCas9-VP64 =5459, 3016, 1337
 Hover name
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL

--> 37°C/ ~10 min.

CONCLUSIONS

  • Success! Keep clone #1 (lane 1). Continue to next step: histone insertions


Rene - PCR libraries

  • Inoculate 200 μL mini-cultures with individual colonies picked from PCR cloning plates with sterile micropipette tips
  1. Luc14g034: 192 colonies, 2 96-well plates (A and B)
  2. Gal4EEDg034: 192 colonies, 2 96-well plates (A and B)
  • Seal with parafilm, grow at 37°C with shaking
  • Spot cultures from 96-well plates onto LB agar amp (100μg/mL) using multichannel pipettor
    • Transferred 2 μL from each well to a 6x8 "array"
    • Each 96-well set was spotted onto two plates (A1-G1, A7-G7)
    • 8 plates total



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