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
1. E. coli DH5α-T NLS-HIS-STOP/size, 25 ng +V0120 (X/P)/3200, 14.1ng NLS-HIS-STOP/C-His 3:1 No Colonies
2. E. coli BL-21 NLS-HIS-STOP/size, 25 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, 25 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, 7.2 ng + V0120 (X/P)/3200, 14.1ng NLS-HIS-STOP/C-His 3:1
5. E. coli BL-21 NLS-HIS-STOP/size, 7.2 ng + V0120 (X/P)/3200, 14.1ng NLS-HIS-STOP/C-His 3:1
6. E. coli BL-21 NLS-HIS-STOP/size, 7.2 ng + V0120 (X/P)/3200, 14.1ng NLS-HIS-STOP/C-His 3:1
7. V0120 (X/P)/3200)/ 15 ng (Control Plate)
  • Reactions 4, 5, 6 the annealing process of the oligos were done in 94°C.
  • 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 6 7
Insert DNA 0.20 0.20 2.05 0.20 0.5 0.70 ---
Vector DNA 3.50 3.50 3.50 3.50 3.50 3.50 3.50
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.2 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O 0 .30 0.30 0.25 0.30 0.00 --- 0.50
  10.00 μL 10.00 μL 10μl 10μL 10μL 10μL 10μL
  • The incubation time for the ligation process was 30min at room temperature.
  • Reaction number 1, 30 μL DH5α-Turbo; ice 5 min.; plate on amp agar
  • Reaction number 2, 3, 4, 5, 6, 7 30μL BL21 in 2.0 mL tubes; ice 2 min.; 42°C 45 sec.; add 800 μL SOC medium; shake @ 37°C 30 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar
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