User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/04/18: Difference between revisions
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''' | '''Building a Reporter Gene to Determine Chromatin Protein Function''' | ||
# | |||
*Ligation of KAH182 = BBa_S04739 and KAH201 = BBa_S04745 and cloning in DH5α-T. | |||
*Assembly strategy: The backbone KAH201(S/P)/3403 , the insert is KAH182(X/P)/1675. | |||
* Ligations | |||
{| {{table}} cellspacing="3" <!-- Ligations table --> | |||
|- bgcolor=#cfcfcf | |||
| Ligation || <font color="blue"><u></font> | |||
|- | |||
| 1. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng || <font color="blue">KAH182/KAH201 2:1 No Colonies</font> | |||
|- | |||
| 2. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng || <font color="blue">KAH182/KAH201 3:1 No Colonies</font> 3:1 1 Colony </font> | |||
|- | |||
| 3. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng || <font color="blue">KAH182/KAH201 4:1 Two Colonies</font> | |||
|- | |||
| 4. KAH201(S/P)/size, 36.29ng (Control Plate)|| | |||
|} | |||
* Calculations are for the ng of insert we need to get a 2:1, 3:1 and 4:1 ratios of insert molecules to 50 ng vector molecules | |||
{| {{table}} cellspacing="3" <!-- Ligation rxn table --> | |||
| || 1 (2:1) || 2 (3:1) || 3 (4:1) || 4 (- Ctrl) | |||
|- | |||
| Insert DNA || 3.11 || 4.66 || 6.21 || --- | |||
|- | |||
| Vector DNA || 1.37 || 1.37 || 1.37 || 1.37 | |||
|- | |||
| 2x lgn buf (Roche) || 5.48 || 7.03 || 8.58 || 5.0 | |||
|- | |||
| T4 ligase (NEB) || 1.0 || 1.0 || 1.0 || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || 0.00 || 0.00 || 0.00 || || 2.63 | |||
|- | |||
| || 10.96 μL || 14.06 μL || 17.16μl || 10μL | |||
|} | |||
* The incubation time for the ligation process was 30min at room temperature. | |||
* Fast transformation, 30 min on ice. |
Revision as of 21:55, 18 April 2013
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04/18/2013Building a Reporter Gene to Determine Chromatin Protein Function
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