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

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| Ligation || <font color="blue"><u></font>
| Ligation || <font color="blue"><u></font>
|-
|-
-
| 1. E. coli DH5α-T NLS-HIS-STOP/size, 20 ng +V0120 (X/P)/3200, 14.1ng || <font color="blue">NLS-HIS-STOP/C-His 3:1 No Colonies</font>  
+
| 1. E. coli DH5α-T NLS-HIS-STOP/size, 25 ng +V0120 (X/P)/3200, 14.1ng || <font color="blue">NLS-HIS-STOP/C-His 3:1 No Colonies</font>  
|-
|-
-
| 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>  
+
| 2. E. coli BL-21  NLS-HIS-STOP/size, 25 ng + V0120 (X/P)/3200, 14.1ng || <font color="blue">NLS-HIS-STOP/C-His 3:1 No Colonies</font>  
|-
|-
-
| 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>  
+
| 3. E. coli BL-21 NLS-HIS-STOP/size, 25 ng + V0120 (X/P)/3200, 14.1ng || <font color="blue">NLS-HIS-STOP/C-His 2:1 No Colonies</font>  
|-
|-
-
| 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>  
+
| 4. E. coli BL-21  NLS-HIS-STOP/size, --- ng + V0120 (X/P)/3200, 14.1ng || <font color="blue">NLS-HIS-STOP/C-His 3:1 </font>  
|-
|-
| 5. V0120 (X/P)/3200)/ 15 ng (Control Plate)||  
| 5. V0120 (X/P)/3200)/ 15 ng (Control Plate)||  
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* The incubation time for the ligation process was 30min at room temperature.
* The incubation time for the ligation process was 30min at room temperature.
-
* Fast transformation, 30 min on ice.
+
* 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 90 sec.; add 800 μL SOC medium; shake @ 37°C 25 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar

Revision as of 16:49, 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, 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, --- 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 0.20 0.20 2.05 --- ---
Vector DNA 3.50 3.50 3.50 --- 3.50
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0
dH2O 0 .30 0.30 0.25 0.03 0.50
  10.00 μL 10.00 μL 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 90 sec.; add 800 μL SOC medium; shake @ 37°C 25 min.; pellet @ top speed 3 min.; resuspend in 100 μL amp liq. medium; plate on amp agar

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