User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/10: Difference between revisions
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''' | '''Site Directed Mutagenesis-Removing the BSMBI cut site from BD002 reporter gene''' | ||
# | |||
# Template strands are about 7kb for BD002. | |||
# Forward primer: 0.15 mg and 32.6 nm, add 326μL dH2O to make 100μM and then diluted to 10μM concentration. | |||
# Reverse primer: 0.16mg and 34.1 nm, add 341μL dH2O to make 100μM and then diluted to 10μM concentration. | |||
# DNA template (BD002) miniprep concentration: 116ng/μL. Accuracy of the BD002 where checked with SapI and NotI cut. | |||
# Serial dilution of template to: 1. 100 ng/μL 2. 80 ng/μL 3. 10 ng/μL 4. 1 ng/μL 5. 200 pg/μL | |||
{| class="wikitable" border=1 cellpadding="7" cellspacing="0" | |||
|- | |||
| Reagents || 1 || 2 || 3 || 4 || 5 | |||
|- | |||
| Plasmid DNA || 0.2 μL || 0.5 μL || 0.5 μL || 0.5 μL || 1 μL | |||
|- | |||
| primer 1 (10 μM, 125 ng) || 1 μL || 1 μL || 1 μL || 1 μL || 1 μL | |||
|- | |||
| primer 2 (10 μM, 125 ng) || 1 μL || 1 μL || 1 μL || 1 μL || 1 μL | |||
|- | |||
| 2X GOTAG (PCR Master Mix)|| 25μL || 25μL || 25μL || 25μL || 25μL | |||
|- | |||
| dH<sub>2</sub>O || 22.8 μL || 22.5 μL || 22.5 μL || 22.5 μL || 22 μL | |||
|- | |||
| Total || 50 μL || 50 μL || 50 μL || 50 μL || 50 μL | |||
|} | |||
*No control reaction, I will test the accuracy of the mutagenesis with BSMBI restriction enzyme. | |||
* '''Thermal Cycling''' | |||
** 95°C/ 30 sec | |||
** [95°C/ 30 sec; 55°C/ 1 min; 72°C/ '''6''' min (1 min/kb plasmid length)]x30 cycle | |||
** 4°C, ∞ | |||
* Run 5 μL of each PCR reaction on 1% agarose gel. | |||
[[Image:BD002-SD-SAPI.png |frame|none|alt=Alt text|Columns 1,3 amplified show linearized BD002 ~6300 bp.]] | |||
* '''DNA Purification''' Using the GenElute™ PCR Clean-Up Kit for rapid purification of double-stranded PCR amplification products. | |||
** The purified result: Column1: 100ng/μL and Column 3: 28ng/μL. | |||
* Cutting the SAPI sites to make the sticky ends for ligation: | |||
{| {{table}} cellspacing="3" width=350px | |||
| Amplified DNA (500ng) || 5.00 μl || 17.00 μL | |||
|- | |||
| SAPI || 1.0 μl || 1.0 μL | |||
|- | |||
| 10x buffer || 5.00 μl || 5.00 μL | |||
|- | |||
| dH<sub>2</sub>O || 39.00 μl || 27.00 μl | |||
|- | |||
| Total || 50.0 μl || 50.0 μl | |||
|- | |||
| rowspan | Incubate at 37°C overnight. | |||
|} | |||
* '''Restriction enzyme deactivation''' Incubate the tubes at 80°C for 10 minutes and cool down gradually in room temperature. | |||
*'''Ligate The Sticky Ends to Make Circular DNA''' | |||
{| {{table}} cellspacing="7" width=650px | |||
| DNA Conc. || 10ng || 20ng || 30ng || 50ng || 50ng (Ctrl) | |||
|- | |||
| DNA μL || 1μL || 2μL || 3μL || 5μL || 5μL | |||
|- | |||
| T4 Ligase || 1.0 μl || 1.0μL || 1.0μL || 1.0μL || 0.0μL | |||
|- | |||
| 2X Roche Buffer|| 5.00 μl || 5.00 μL || 5.00 μL || 5.00 μL || 5.00 μL | |||
|- | |||
| dH<sub>2</sub>O || 5.00 μl || 5.00 μl || 5.00 μL || 6.00 μL || 0.00 μL | |||
|- | |||
| Total || 10.0 μl || 10.0 μl || 10.0 μl || 10.0 μl || 10.0 μl | |||
|- | |||
| Incubate at room temperature for 30 minutes. | |||
|- | |||
| '''Transformation result''' || No colony || No colony || No colony || <span style="color:#FF0000">Two Colonies</span>|| <span style="color:#FF0000">No Colony</span> | |||
|} | |||
* Long transformation protocol in BL-21. | |||
* Grow on AMP agar plates and incubate overnight at 37°C. | |||
'''Mutagenesis Confirmation''' | |||
* Grow the colonies in LB broth for 6 hrs. | |||
* Miniprep the plasmids, final concentration: '''491ng/μL''' | |||
[[Image:BD002-No BSMBI-Gel Proof.png|thumb|280px| Column 1 and 4 look the same, which shows that no BSMBI cut site is recognized in column 1, Colom 5 and 3 look the same which shows only SpeI works and linearizes the plasmid in column 5. Linearize plasmid size: ~6300 bps]] | |||
{| {{table}} ; style="text-align:center; width:550px; height:200px;" | |||
| Plasmid DNA (BD002)|| 1 (4.0 μl) || 2 (4.0 μl)|| 3 (4.0 μl)|| 4 (4.0 μl)|| 5 (4.0 μl) | |||
|- | |||
| BSMBI || 1.0 μl || --- || --- || --- || 1.0 μl | |||
|- | |||
| NotI || --- || 1.0 μl || 1.0 μl || --- || 1.0 μl | |||
|- | |||
| SpeI || --- || 1.0 μl || --- || --- || --- | |||
|- | |||
| 10x FastDigest buffer + green loading dye || 1.5 μl || 1.5 μl || 1.5 μl || 1.5 μl || 1.5 μl | |||
|- | |||
| dH<sub>2</sub>O || 9.5 μl || 7.5μL || 9.5μL|| 10.5μL || 7.5μL | |||
|- | |||
| Total || 15.0 μl || 15.0 μl || 15.0 μl || 15.0 μl|| 15.0 μl | |||
|- | |||
| Incubate at 37°C for 30 minutes. | |||
|} | |||
# Make the (1%) agarose glee and add 15μl of restricted vector in one well and 10 μl of ladder. | |||
'''Sequencing Analysis''' | |||
Mutated cut site is marked with red. | |||
[[Image:Sequencing Result.png | 600px]] |
Revision as of 14:04, 4 November 2013
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10/10/12Site Directed Mutagenesis-Removing the BSMBI cut site from BD002 reporter gene
Mutagenesis Confirmation
Sequencing Analysis Mutated cut site is marked with red. |