User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/01/12: Difference between revisions
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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 --> | ||
|- bgcolor=#cfcfcf | |- bgcolor=#cfcfcf | ||
| Ligation || <font color="blue"><u> | | Ligation || <font color="blue"><u></font> | ||
|- | |- | ||
| 1. E. coli | | 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 | | 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. C-His(E/P)/ | | 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> | ||
|- | |||
| 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 --> | {| {{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. |
Revision as of 14:52, 12 January 2013
PcTF Subcloning in E. coli | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||
01/12/2013Ligation of KAH111, KAH112 and C-His vector in DH5α-T and BL21
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