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"| | |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= | =01/12/2013= | ||
Ligation of KAH111, KAH112 and C-His vector in DH5α-T and BL21 | 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 | * 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 | * Ligations | ||
{| {{table}} cellspacing="3" <!-- Ligations table --> | {| {{table}} cellspacing="3" <!-- Ligations table --> | ||
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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 --> | ||
| || 1 || 2 || 3 || | | || 1 || 2 || 3 || 4 || 5 | ||
|- | |- | ||
| Insert DNA || | | Insert DNA || 2.34 || 2.34 || 2.05 || 2.05 || --- | ||
|- | |- | ||
| Vector DNA || | | Vector DNA || 1.92 || 1.92 || 1.92 || 1.92 || 1.92 | ||
|- | |- | ||
| 2x lgn buf (Roche) || | | 2x lgn buf (Roche) || 5.26 || 5.26 || 5.0 || 5.0 || 5.0 | ||
|- | |- | ||
| T4 ligase (NEB) || 1.0 || 1.0 || 1.0 || | | T4 ligase (NEB) || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || 0 || 0 || 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
PcTF Subcloning in E. coli | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||
01/12/2013Ligation of KAH111, KAH112 and C-His vector in DH5α-T and BL21
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