User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/01/24

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(01/24/2013)
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NLS-6His-STOP-F1 CTAGAcccaagaaaaagcgcaaggtacaccatcaccaccatcacgcgtaaagctgagACTAGTAGCGGCCGCTGCA 76
NLS-6His-STOP-F1 CTAGAcccaagaaaaagcgcaaggtacaccatcaccaccatcacgcgtaaagctgagACTAGTAGCGGCCGCTGCA 76
 +
NLS-6His-STOP-R1 GCGGCCGCTACTAGTctcagctttacgcgtgatggtggtgatggtgtaccttgcgctttttcttgggT 68
NLS-6His-STOP-R1 GCGGCCGCTACTAGTctcagctttacgcgtgatggtggtgatggtgtaccttgcgctttttcttgggT 68
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Heat at 100°C for 5 min., remove the entire heat block or water bath from the heat source, and allow to cool slowly to room temperature.<br>
Heat at 100°C for 5 min., remove the entire heat block or water bath from the heat source, and allow to cool slowly to room temperature.<br>
--> *10x annealing buffer: 1 M NaCl; 100 mM Tris-HCl, pH 7.4
--> *10x annealing buffer: 1 M NaCl; 100 mM Tris-HCl, pH 7.4
 +
 +
 +
Ligation of NLS-HIS-STOP and V0120 vector in DH5α-T and BL21
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* V0120 cut with X/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 DH5α-T NLS-HIS-STOP/size, 20 ng +V0120 (X/P)/3200, 14.1ng || <font color="blue">KNLS-HIS-STOP/C-His 3:1 No Colonies</font>
 +
|-
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| 2. E. coli BL-21  NLS-HIS-STOP/size, 16 ng + V0120 (X/P)/3200, 14.1ng || <font color="blue">NLS-HIS-STOP/C-His 3:1 No Colonies</font>
 +
|-
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| 3. E. coli BL-21 NLS-HIS-STOP/size, 22.8 ng + V0120 (X/P)/3200, 14.1ng || <font color="blue">NLS-HIS-STOP/C-His 2:1 No Colonies</font>
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|-
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| 4. E. coli BL-21  NLS-HIS-STOP/size, 20 ng + V0120 (X/P)/3200, 14.1ng || <font color="blue">NLS-HIS-STOP/C-His 3:1 </font>
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|-
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| 5. V0120 (X/P)/3200)/ 15 ng (Control Plate)||
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|}
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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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{| {{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.

Revision as of 16:39, 25 January 2013

PcTF Subcloning in E. coli Main project page
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01/24/2013

'Making Standardized DNA Part (NLS-HIS-STOP)'

NLS-6His-STOP-F1 CTAGAcccaagaaaaagcgcaaggtacaccatcaccaccatcacgcgtaaagctgagACTAGTAGCGGCCGCTGCA 76

NLS-6His-STOP-R1 GCGGCCGCTACTAGTctcagctttacgcgtgatggtggtgatggtgtaccttgcgctttttcttgggT 68

Set up an annealing reaction as follows:

Sense oligo 1 (100 μM) 3.0 μl
Anti-sense oligo (100 μM) 3.0 μl
10x annealing buffer* 2.0 μl
dH2O 12.0 μl
  20 μl

Heat at 100°C for 5 min., remove the entire heat block or water bath from the heat source, and allow to cool slowly to room temperature.
--> *10x annealing buffer: 1 M NaCl; 100 mM Tris-HCl, pH 7.4


Ligation of NLS-HIS-STOP and V0120 vector in DH5α-T and BL21

  • V0120 cut with X/P and purified from the gel.
  • Ligations
Ligation </font>
1. E. coli DH5α-T NLS-HIS-STOP/size, 20 ng +V0120 (X/P)/3200, 14.1ng KNLS-HIS-STOP/C-His 3:1 No Colonies
2. E. coli BL-21 NLS-HIS-STOP/size, 16 ng + V0120 (X/P)/3200, 14.1ng NLS-HIS-STOP/C-His 3:1 No Colonies
3. E. coli BL-21 NLS-HIS-STOP/size, 22.8 ng + V0120 (X/P)/3200, 14.1ng NLS-HIS-STOP/C-His 2:1 No Colonies
4. E. coli BL-21 NLS-HIS-STOP/size, 20 ng + V0120 (X/P)/3200, 14.1ng NLS-HIS-STOP/C-His 3:1
5. V0120 (X/P)/3200)/ 15 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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