User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/01/24: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">PcTF Subcloning in E. coli</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">PcTF Subcloning in E. coli</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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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 of NLS-HIS-STOP and V0120 vector in DH5α-T and BL21 | Ligation of NLS-HIS-STOP and V0120 vector in DH5α-T and BL21 | ||
* V0120 cut with X/P and purified from the gel. | * V0120 cut with X/P and purified from the gel. | ||
<div class="floatright">[[Image: V0120(X-P).png ]]<br>V0120 Vector cut with Xba1 and Pst1 | |||
</div> | |||
* Ligations | * Ligations | ||
{| {{table}} cellspacing="3" <!-- Ligations table --> | {| {{table}} cellspacing="3" <!-- Ligations table --> | ||
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| Ligation || <font color="blue"><u></font> | | Ligation || <font color="blue"><u></font> | ||
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| 1. E. coli DH5α-T NLS-HIS-STOP/size, | | 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> | ||
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| 2. E. coli BL-21 NLS-HIS-STOP/size, | | 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> | ||
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| 3. E. coli BL-21 NLS-HIS-STOP/size, | | 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> | ||
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| 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 No Colonies </font> | ||
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| 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 No Colonies</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 No Colonies</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 || | | Insert DNA || 0.20 || 0.20 || 2.05 || 0.20 || 0.5 ||0.70 ||--- | ||
|- | |- | ||
| Vector DNA || | | Vector DNA || 3.50 || 3.50 || 3.50 || 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 || 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 || 0 || 0. | | dH<sub>2</sub>O || 0 .30|| 0.30 || 0.25 || 0.30 || 0.00 || --- || 0.50 | ||
|- | |- | ||
| || 10. | | || 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 2, 3, 4, 5, 6, 7 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 |
Latest revision as of 22:23, 26 September 2017
PcTF Subcloning in E. coli | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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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