User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/04/18: Difference between revisions

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=Date=
=04/18/2013=


'''List title'''
'''Building a Reporter Gene to Determine Chromatin Protein Function'''
# List items
 
*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 -->
| &nbsp;            || 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
|-
| &nbsp;            || 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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04/18/2013

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
Ligation
1. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng KAH182/KAH201 2:1 No Colonies
2. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng KAH182/KAH201 3:1 No Colonies 3:1 1 Colony
3. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng KAH182/KAH201 4:1 Two Colonies
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
  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
dH2O 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.
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