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

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=01/24/2013=
=01/24/2013=


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


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
* V0120 cut with X/P and purified from the gel. 
* Ligations
{| {{table}} cellspacing="3" <!-- Ligations table -->
|- bgcolor=#cfcfcf
| Ligation || <font color="blue"><u></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, 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, 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, --- 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)||
|}
* Calculations are for the ng of insert we need to get a 3:1 ratio of insert molecules to 50 ng vector molecules
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 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
|-
| dH<sub>2</sub>O    || 0 .30|| 0.30 || 0.25  || 0.03 || 0.50
|-
| &nbsp;            || 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

Revision as of 13:49, 25 January 2013

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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, --- 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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