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 | ||
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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"> | | 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> | ||
|- | |- | ||
| 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, 16 ng + V0120 (X/P)/3200, 14.1ng || <font color="blue">NLS-HIS-STOP/C-His 3:1 No Colonies</font> | ||
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| || 1 || 2 || 3 || 4 || 5 | | || 1 || 2 || 3 || 4 || 5 | ||
|- | |- | ||
| Insert DNA || | | Insert DNA || 0.20 || 0.20 || 2.05 || --- || --- | ||
|- | |- | ||
| Vector DNA || | | Vector DNA || 3.50 || 3.50 || 3.50 || --- || 3.50 | ||
|- | |- | ||
| 2x lgn buf (Roche) || 5. | | 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 | | T4 ligase (NEB) || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || 0 || 0 || 0. | | dH<sub>2</sub>O || 0 .30|| 0.30 || 0.25 || 0.03 || 0.50 | ||
|- | |- | ||
| || 10. | | || 10.00 μL || 10.00 μL || 10μl || 10μL || 10μL | ||
|} | |} | ||
* 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. | * Fast transformation, 30 min on ice. |
Revision as of 13:43, 25 January 2013
PcTF Subcloning in E. coli | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
01/24/2013Making 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:
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.
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