Difference between revisions of "User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/01/24"
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| 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> | | 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, | + | | 4. E. coli BL-21 NLS-HIS-STOP/size, 7.2 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. E. coli BL-21 NLS-HIS-STOP/size, 7.2 ng + V0120 (X/P)/3200, 14.1ng || <font color="blue">NLS-HIS-STOP/C-His 3:1 </font> |
| + | |- | ||
| + | | 6. E. coli BL-21 NLS-HIS-STOP/size, 7.2 ng + V0120 (X/P)/3200, 14.1ng || <font color="blue">NLS-HIS-STOP/C-His 3:1 </font> | ||
| + | |- | ||
| + | | 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 | * 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 --> | {| {{table}} cellspacing="3" <!-- Ligation rxn table --> | ||
| − | | || 1 || 2 || 3 || 4 || 5 | + | | || 1 || 2 || 3 || 4 || 5 || 6 || 7 |
|- | |- | ||
| − | | Insert DNA || 0.20 || 0.20 || 2.05 || | + | | Insert DNA || 0.20 || 0.20 || 2.05 || 0.20 || 0.5 ||0.70 ||--- |
|- | |- | ||
| − | | Vector DNA || 3.50 || 3.50 || 3.50 || | + | | 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 | + | | 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 | + | | T4 ligase (NEB) || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 |
|- | |- | ||
| − | | dH<sub>2</sub>O || 0 .30|| 0.30 || 0.25 || 0. | + | | dH<sub>2</sub>O || 0 .30|| 0.30 || 0.25 || 0.30 || 0.00 || --- || 0.50 |
|- | |- | ||
| − | | || 10.00 μL || 10.00 μL || 10μl || 10μL || 10μL | + | | || 10.00 μL || 10.00 μL || 10μl || 10μL || 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. | ||
* Reaction number 1, 30 μL DH5α-Turbo; ice 5 min.; plate on amp agar | * 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 | + | * 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 |
Revision as of 16:34, 25 January 2013
 PcTF Subcloning in E. coli
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<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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