User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/01/12

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=Date=
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=01/12/2013=
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Ligation of KAH111, KAH112 and C-His vector in DH5α-T and BL21
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* 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. 
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* Ligations
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{| {{table}} cellspacing="3" <!-- Ligations table -->
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|- bgcolor=#cfcfcf
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| Ligation || <font color="blue"><u></font>
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|-
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| 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>
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|-
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| 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>
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|-
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| 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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|-
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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>
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|-
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| 5. C-His(E/P)/ 26 ng (Control Plate)||
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|}
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'''List title'''
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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
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# List items
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{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
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| &nbsp;            || 1    || 2  || 3  || 4 || 5
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|-
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| Insert DNA        || 2.34 || 2.34 || 2.05  || 2.05 || ---
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|-
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| Vector DNA        || 1.92  || 1.92 || 1.92  || 1.92 || 1.92
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|-
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| 2x lgn buf (Roche) || 5.26  || 5.26 || 5.0 || 5.0 || 5.0
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|-
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| T4 ligase (NEB)    || 1.0  || 1.0  || 1.0 || 1.0 || 1.0
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|-
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| dH<sub>2</sub>O    || 0 || 0 || 0.03  || 0.03 || 2.08
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|-
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| &nbsp;            || 10.52 μL || 10.52 μL || 10μl || 10μL || 10μL
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|}
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* The incubation time for the ligation process was 30min at room temperature.
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* Fast transformation, 30 min on ice.

Current revision

PcTF Subcloning in E. coli Main project page
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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
Ligation </font>
1. E. coli BL-21 KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng KAH111/C-His 3:1 No Colonies
2. E. coli DH5α-T KAH111(E/P)/size, 16 ng + C-His(E/P)/3600, 26ng KAH111/C-His 3:1 No Colonies
3. E. coli BL-21 KAH112(E/P)/size, 22.8 ng + C-His(E/P)/3600, 26ng KAH112/C-His 3:1 No Colonies
4. E. coli DH5α-T KAH111(E/P)/size, 20 ng + C-His(E/P)/3600, 26ng KAH112/C-His 3:1 No Colonies
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
  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
dH2O 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.

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