User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/01/12: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">PcTF Subcloning in E. coli</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">PcTF Subcloning in E. coli</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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* Calculations are for the ng of insert we need to get a 3:1 ratio of insert molecules to 50 ng vector molecules
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2  || 3  || 4 || 5
| &nbsp;            || 1    || 2  || 3  || 4 || 5
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| dH<sub>2</sub>O    || 0 || 0 || 0.03  || 0.03 || 2.08
| dH<sub>2</sub>O    || 0 || 0 || 0.03  || 0.03 || 2.08
|-
|-
| &nbsp;            || 10.52 μL || 10.52 μL || 10μl || 10μL  
| &nbsp;            || 10.52 μL || 10.52 μL || 10μl || 10μL || 10μL  
|}
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* The incubation time for the ligation process was 30min at room temperature.
* Fast transformation, 30 min on ice.

Latest revision as of 22:21, 26 September 2017

PcTF Subcloning in E. coli Main project page
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01/12/2013

Ligation of KAH111, KAH112 and C-His vector in DH5α-T and BL21

  • The 4 μL of C-His Vector from stock is diluted with 16 μL of nuclease free water, then cut with E/P and purified from the gel.
  • Ligations
Ligation
1. E. coli BL-21 KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng KAH111/C-His 3:1 No Colonies
2. E. coli DH5α-T KAH111(E/P)/size, 16 ng + C-His(E/P)/3600, 26ng KAH111/C-His 3:1 No Colonies
3. E. coli BL-21 KAH112(E/P)/size, 22.8 ng + C-His(E/P)/3600, 26ng KAH112/C-His 3:1 No Colonies
4. E. coli DH5α-T KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng KAH112/C-His 3:1 No Colonies
5. C-His(E/P)/ 26 ng (Control Plate)
  • Calculations are for the ng of insert we need to get a 3:1 ratio of insert molecules to 50 ng vector molecules
  1 2 3 4 5
Insert DNA 2.34 2.34 2.05 2.05 ---
Vector DNA 1.92 1.92 1.92 1.92 1.92
2x lgn buf (Roche) 5.26 5.26 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0
dH2O 0 0 0.03 0.03 2.08
  10.52 μL 10.52 μL 10μl 10μL 10μL
  • The incubation time for the ligation process was 30min at room temperature.
  • Fast transformation, 30 min on ice.
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