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

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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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