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, 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, 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
|
PcTF Subcloning in E. coli
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