User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/01/24: Difference between revisions
From OpenWetWare
(→Date) |
|||
Line 12: | Line 12: | ||
NLS-6His-STOP-F1 CTAGAcccaagaaaaagcgcaaggtacaccatcaccaccatcacgcgtaaagctgagACTAGTAGCGGCCGCTGCA 76 | NLS-6His-STOP-F1 CTAGAcccaagaaaaagcgcaaggtacaccatcaccaccatcacgcgtaaagctgagACTAGTAGCGGCCGCTGCA 76 | ||
NLS-6His-STOP-R1 GCGGCCGCTACTAGTctcagctttacgcgtgatggtggtgatggtgtaccttgcgctttttcttgggT 68 | NLS-6His-STOP-R1 GCGGCCGCTACTAGTctcagctttacgcgtgatggtggtgatggtgtaccttgcgctttttcttgggT 68 | ||
Line 30: | Line 31: | ||
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, 20 ng +V0120 (X/P)/3200, 14.1ng || <font color="blue">KNLS-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> | |||
|- | |||
| 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> | |||
|- | |||
| 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> | |||
|- | |||
| 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 --> | |||
| || 1 || 2 || 3 || 4 || 5 | |||
|- | |||
| Insert DNA || 2.34 || 2.34 || 2.05 || 2.05 || --- | |||
|- | |||
| Vector DNA || 1.92 || 1.92 || 1.92 || 1.92 || 1.92 | |||
|- | |||
| 2x lgn buf (Roche) || 5.26 || 5.26 || 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 || 0 || 0.03 || 0.03 || 2.08 | |||
|- | |||
| || 10.52 μL || 10.52 μL || 10μl || 10μL || 10μL | |||
|} | |||
* The incubation time for the ligation process was 30min at room temperature. | |||
* Fast transformation, 30 min on ice. |
Revision as of 13:39, 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> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
01/24/2013'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:
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.
|