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="5" cellspacing="0"
{| class="wikitable" border=1 cellpadding="7" cellspacing="0"
|-
|-
| Reagents || 1 || 2 || 3 || 4 || 5
| Reagents || 1 || 2 || 3 || 4 || 5
Line 39: Line 39:
** [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 10% agarose gel.
* Run 5 μL of each PCR reaction on 1% agarose gel.


[[Image:BD002-SD-SAPI.png]]
[[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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10/10/12

Site Directed Mutagenesis-Removing the BSMBI cut site from BD002 reporter gene

  1. Template strands are about 7kb for BD002.
  2. Forward primer: 0.15 mg and 32.6 nm, add 326μL dH2O to make 100μM and then diluted to 10μM concentration.
  3. Reverse primer: 0.16mg and 34.1 nm, add 341μL dH2O to make 100μM and then diluted to 10μM concentration.
  4. DNA template (BD002) miniprep concentration: 116ng/μL. Accuracy of the BD002 where checked with SapI and NotI cut.
  5. 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
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
dH2O 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.
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:
Amplified DNA (500ng) 5.00 μl 17.00 μL
SAPI 1.0 μl 1.0 μL
10x buffer 5.00 μl 5.00 μL
dH2O 39.00 μl 27.00 μl
Total 50.0 μl 50.0 μl
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
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
dH2O 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 Two Colonies No Colony
  • 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
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
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
dH2O 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.
  1. 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.

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