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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"| | |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]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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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 | |||
{| {{table}} cellspacing="3" <!-- Ligations table --> | |||
|- bgcolor=#cfcfcf | |||
| Ligation || <font color="blue"><u></font> | |||
|- | |||
| 1. E. coli BL-21 KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng || <font color="blue">KAH111/C-His 3:1 No Colonies</font> | |||
|- | |||
| 2. E. coli DH5α-T KAH111(E/P)/size, 16 ng + C-His(E/P)/3600, 26ng || <font color="blue">KAH111/C-His 3:1 No Colonies</font> | |||
|- | |||
| 3. E. coli BL-21 KAH112(E/P)/size, 22.8 ng + C-His(E/P)/3600, 26ng || <font color="blue">KAH112/C-His 3:1 No Colonies</font> | |||
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| 4. E. coli DH5α-T KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng || <font color="blue">KAH112/C-His 3:1 No Colonies</font> | |||
|- | |||
| 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 | |||
{| {{table}} cellspacing="3" <!-- Ligation rxn table --> | |||
| || 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 | |||
|- | |||
| dH<sub>2</sub>O || 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. |
Latest revision as of 22:21, 26 September 2017
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01/12/2013Ligation of KAH111, KAH112 and C-His vector in DH5α-T and BL21
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