User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/10/10: Difference between revisions
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# 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 | # 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=" | {| class="wikitable" border=1 cellpadding="7" cellspacing="0" | ||
|- | |- | ||
| Reagents || 1 || 2 || 3 || 4 || 5 | | Reagents || 1 || 2 || 3 || 4 || 5 | ||
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** [95°C/ 30 sec; 55°C/ 1 min; 72°C/ '''6''' min (1 min/kb plasmid length)]x30 cycle | ** [95°C/ 30 sec; 55°C/ 1 min; 72°C/ '''6''' min (1 min/kb plasmid length)]x30 cycle | ||
** 4°C, ∞ | ** 4°C, ∞ | ||
* Run 5 μL of each PCR reaction on | * 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 BD002 ~6300 bp.]] | [[Image:BD002-SD-SAPI.png |frame|none|alt=Alt text|Columns 1,3 amplified show BD002 ~6300 bp.]] | ||
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*'''Ligate The Sticky Ends to Make Circular DNA''' | *'''Ligate The Sticky Ends to Make Circular DNA''' | ||
{| {{table}} cellspacing=" | {| {{table}} cellspacing="7" width=650px | ||
| DNA Conc. || 10ng || 20ng || 30ng || 50ng || 50ng (Ctrl) | | DNA Conc. || 10ng || 20ng || 30ng || 50ng || 50ng (Ctrl) | ||
|- | |- | ||
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* Miniprep the plasmids, final concentration: '''491ng/μL''' | * Miniprep the plasmids, final concentration: '''491ng/μL''' | ||
[[Image: | [[Image:BD002-No BSMBI-Gel Proof.png|thumb|450px| 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}} | {| {{table}} ; style="text-align:center; width:550px; height:200px;" | ||
| Plasmid DNA (BD002) (4.0 μl | | 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 || | | BSMBI || 1.0 μl || --- || --- || --- || 1.0 μl | ||
|- | |- | ||
| | | NotI || --- || 1.0 μl || 1.0 μl || --- || 1.0 μl | ||
|- | |- | ||
| 10x FastDigest buffer + green loading dye || 1.5 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 | | 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 | | 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. | # Make the (1%) agarose glee and add 15μl of restricted vector in one well and 10 μl of ladder. |
Revision as of 09:49, 16 October 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> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
10/10/12Site Directed Mutagenesis-Removing the BSMBI cut site from BD002 reporter gene
Mutagenesis Confirmation
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