Difference between revisions of "Team:Freiburg/Protocols/Gibson Assembly"

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<div class="content_box">
 
<div class="content_box">
 
<h1 class="sectionedit1"><a name="gibson_assembly" id="gibson_assembly">Gibson Assembly</a></h1>
 
<h1 class="sectionedit1"><a name="gibson_assembly" id="gibson_assembly">Gibson Assembly</a></h1>
 
<div class="level1">
 
<div class="level1">
 +
<p>
 +
Gibson Assembly is rather novel method for assembling DNA fragments with overlapping overhangs. The operating mode of this method is devided into three major parts:
 +
 +
<ol>
 +
<li>An exonuclease removes bases from the 5' end of each DNA strand.</li>
 +
<li>Complementary regions of different DNA strands can anneal and a polymerase fills up the gaps.</li>
 +
<li>The fragments are ligated together.</li>
 +
</ol>
 +
<br>
 +
To enable these three steps, DNA strands with compatible ends of about 32 bp are needed. Those can either be incorporated by primer overhangs or by gene synthesis.<br>
 +
A 5 µl mix of the DNA parts that are supposed to be assembled is prepared. Its composition is calculated based on the length and concentration of every single fragment. The insert(s) should at least be contained in a 4 - 8 fold molar amount of the antibiotic resistance containing backbone.<br>
 +
</p>
  
 
<p>
 
<p>
−
<strong>Protocol for the assembly of PCR products with overhangs</strong>
+
<h3>Assembly of DNA fragments with overlapping regions</h3>
 
<em>(adapted from AG Weber protocol)</em>
 
<em>(adapted from AG Weber protocol)</em>
 
</p>
 
</p>
  
 
<p>
 
<p>
−
material: Gibson Master Mix Aliquots<br/>
+
Material: Gibson Master Mix Aliquots<br/>
−
 
+
−
time: 90 min<br/>
+
  
 +
Time: 90 min<br/>
 
</p>
 
</p>
 
<hr />
 
<hr />
Line 27: Line 61:
 
</p>
 
</p>
 
<ol>
 
<ol>
−
<li class="level1"><div class="li"> prepare 5µl of DNA-Mix (calculate voulumes using the equations below or use the prepared worksheet: <a href="/igem2015/lib/exe/fetch.php?media=files:protocols:gibson_dna_mix.xlsx" class="media mediafile mf_xlsx" title="files:protocols:gibson_dna_mix.xlsx">Stefan&#039;s ExcelSheet</a></div>
+
<li>Prepare 5 µl of DNA-Mix (calculate voulumes using the equations below or use the <a href="/igem2015/lib/exe/fetch.php?media=files:protocols:gibson_dna_mix.xlsx" class="media mediafile mf_xlsx" title="files:protocols:gibson_dna_mix.xlsx">prepared worksheet</a>.
 
</li>
 
</li>
−
<li class="level1"><div class="li"> add DNA Mix to Gibson Master Mix</div>
+
<li>Add the DNA Mix to Gibson Master Mix.
 
</li>
 
</li>
−
<li class="level1"><div class="li"> incubate on RT for 5 min</div>
+
<li>Incubate for 5 min at RT.
 
</li>
 
</li>
−
<li class="level1"><div class="li"> use 5-7µl for transformation of competent <em>E.coli</em> cells</div>
+
<li>Use 5 - 7 µl for transformation of competent <i>E.coli</i> cells.
 
</li>
 
</li>
 
</ol>
 
</ol>
  
 
</div>
 
</div>
−
 
+
<br>
−
<h5><a name="equations" id="equations">Equations</a></h5>
+
<br>
 +
<h3>Calculation of the DNA mix</h3>
 
<div class="level5">
 
<div class="level5">
  
 
<p>
 
<p>
 +
<li>Backbone:<br>
 
V (Bb) = 12 ng/kb * l (Bb) / c (Bb)<br/>
 
V (Bb) = 12 ng/kb * l (Bb) / c (Bb)<br/>
 +
</li>
  
 +
<li>Insert(s):<br>
 
V (Ins) = 12 ng/kb * l (Ins) * <strong>X</strong> / c (Ins)<br/>
 
V (Ins) = 12 ng/kb * l (Ins) * <strong>X</strong> / c (Ins)<br/>
 +
</li>
  
 
<br/>
 
<br/>
Line 51: Line 90:
 
<strong>X</strong> = Ratio Insert to Backbone<br/>
 
<strong>X</strong> = Ratio Insert to Backbone<br/>
  
−
z.B. <strong>X</strong> = 4, if Ins : Bb = 4 : 1
+
for example: <strong>X</strong> = 4, if Ins : Bb = 4 : 1
 
</p>
 
</p>
  
 
</div>
 
</div>
−
<!-- EDIT1 SECTION "Gibson Assembly" [1-806] -->
 
−
<h1 class="sectionedit2"><a name="gibson_master_mix" id="gibson_master_mix">Gibson master mix</a></h1>
 
−
<div class="level1">
 
  
 +
<!-- EDIT1 SECTION "Gibson Assembly" [1-806] -->
 
<p>
 
<p>
−
 
−
 
 
<body>
 
<body>
−
<h3>A) ISO buffer</h3>
+
 
−
<table class="tabelle">
+
<h3>A) Gibson Assembly Master Mix</h3>
−
<tr><th><center>amount</center></th><th><center>ingredient</center></th><th>remarks</th></tr>
+
−
<tr><td><center>1.5 g</center></td><td><center>PEG-8000</center></td><td></td></tr>
+
−
<tr><td><center>3 ml</center></td><td><center>Tris-HCl (1 M, pH 7.5)</center></td><td>dissolve 12.1 g Tris in 100 ml dH<sub>2</sub>O, adjust pH to 7.5 with conc. HCl</td></tr>
+
−
<tr><td><center>300 µl</center></td><td><center>DTT (1 M)</center></td><td>dissolve 1.54 g DTT in 10 ml dH<sub>2</sub>O</td></tr>
+
−
<tr><td><center>150 µl</center></td><td><center>MgCl<sub>2</sub> (2 M)</center></td><td>dissolve 4.06 g MgCl<sub>2</sub> in 10 ml dH<sub>2</sub>O</td></tr>
+
−
<tr><td><center>300 µl</center></td><td><center>NADNa (100 mM)</center></td><td>dissolve 0.02 g NADNa in 300 µl dH<sub>2</sub>O</td></tr>
+
−
<tr><td><center>4 x 60 µl</center></td><td><center>dNTPs (100 mM, each)</center></td><td></td></tr>
+
−
<tr><td><center>up to 6 ml</center></td><td><center>dH<sub>2</sub>O</center></td><td></td></tr>
+
−
</table>
+
−
<p>aliquot á 350 µl</p>
+
−
<h3>B) Assembly master mix</h3>
+
 
<p><b>Work on ice!</b></p>
 
<p><b>Work on ice!</b></p>
 
<table class="tabelle">
 
<table class="tabelle">
−
<tr><th><center>volume</center></th><th><center>ingredient</center></th></tr>
+
<tr><th><center>Volume</center></th><th><center>Ingredient</center></th></tr>
 
<tr><td><center>690 µl</center></td><td><center>dH<sub>2</sub>O</center></td></tr>
 
<tr><td><center>690 µl</center></td><td><center>dH<sub>2</sub>O</center></td></tr>
 
<tr><td><center>320 µl</center></td><td><center>ISO buffer</center></td></tr>
 
<tr><td><center>320 µl</center></td><td><center>ISO buffer</center></td></tr>
Line 87: Line 111:
 
<p>* dilute 3.2 µl T5 Exonuclease (10 U/µl) in 46.8 µl 1x T5-buffer</p>
 
<p>* dilute 3.2 µl T5 Exonuclease (10 U/µl) in 46.8 µl 1x T5-buffer</p>
 
<p>aliquot á 15 µl</p>
 
<p>aliquot á 15 µl</p>
 +
 +
 +
<h3>B) ISO buffer</h3>
 +
<table class="tabelle">
 +
<tr><th><center>Amount</center></th><th><center>Ingredient</center></th><th>Remarks</th></tr>
 +
<tr><td><center>1.5 g</center></td><td><center>PEG-8000</center></td><td></td></tr>
 +
<tr><td><center>3 ml</center></td><td><center>Tris-HCl (1 M, pH 7.5)</center></td><td>dissolve 12.1 g Tris in 100 ml dH<sub>2</sub>O, adjust pH to 7.5 with conc. HCl</td></tr>
 +
<tr><td><center>300 µl</center></td><td><center>DTT (1 M)</center></td><td>dissolve 1.54 g DTT in 10 ml dH<sub>2</sub>O</td></tr>
 +
<tr><td><center>150 µl</center></td><td><center>MgCl<sub>2</sub> (2 M)</center></td><td>dissolve 4.06 g MgCl<sub>2</sub> in 10 ml dH<sub>2</sub>O</td></tr>
 +
<tr><td><center>300 µl</center></td><td><center>NADNa (100 mM)</center></td><td>dissolve 0.02 g NADNa in 300 µl dH<sub>2</sub>O</td></tr>
 +
<tr><td><center>4 x 60 µl</center></td><td><center>dNTPs (100 mM, each)</center></td><td></td></tr>
 +
<tr><td><center>up to 6 ml</center></td><td><center>dH<sub>2</sub>O</center></td><td></td></tr>
 +
</table>
 +
<p>aliquot á 350 µl</p>
 +
 
</body>
 
</body>
  

Latest revision as of 07:07, 20 November 2015

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Gibson Assembly

Gibson Assembly is rather novel method for assembling DNA fragments with overlapping overhangs. The operating mode of this method is devided into three major parts:

  1. An exonuclease removes bases from the 5' end of each DNA strand.
  2. Complementary regions of different DNA strands can anneal and a polymerase fills up the gaps.
  3. The fragments are ligated together.

To enable these three steps, DNA strands with compatible ends of about 32 bp are needed. Those can either be incorporated by primer overhangs or by gene synthesis.
A 5 µl mix of the DNA parts that are supposed to be assembled is prepared. Its composition is calculated based on the length and concentration of every single fragment. The insert(s) should at least be contained in a 4 - 8 fold molar amount of the antibiotic resistance containing backbone.

Assembly of DNA fragments with overlapping regions

(adapted from AG Weber protocol)

Material: Gibson Master Mix Aliquots
Time: 90 min


  1. Prepare 5 µl of DNA-Mix (calculate voulumes using the equations below or use the prepared worksheet.
  2. Add the DNA Mix to Gibson Master Mix.
  3. Incubate for 5 min at RT.
  4. Use 5 - 7 µl for transformation of competent E.coli cells.


Calculation of the DNA mix

  • Backbone:
    V (Bb) = 12 ng/kb * l (Bb) / c (Bb)
  • Insert(s):
    V (Ins) = 12 ng/kb * l (Ins) * X / c (Ins)

    • X = Ratio Insert to Backbone
    for example: X = 4, if Ins : Bb = 4 : 1

    A) Gibson Assembly Master Mix

    Work on ice!

    Volume
    Ingredient
    690 µl
    dH2O
    320 µl
    ISO buffer
    160 µl
    Taq ligase (NEB, 40 U/µl)
    20 µl
    Q5 Polymerase (NEB, 2 U/µl)
    10 µl
    T5 Exonuclease (NEB, 0.64 U/µl)*

    * dilute 3.2 µl T5 Exonuclease (10 U/µl) in 46.8 µl 1x T5-buffer

    aliquot á 15 µl

    B) ISO buffer

    Amount
    Ingredient
    		Remarks
    1.5 g
    PEG-8000
    3 ml
    Tris-HCl (1 M, pH 7.5)
    		dissolve 12.1 g Tris in 100 ml dH2O, adjust pH to 7.5 with conc. HCl
    300 µl
    DTT (1 M)
    		dissolve 1.54 g DTT in 10 ml dH2O
    150 µl
    MgCl2 (2 M)
    		dissolve 4.06 g MgCl2 in 10 ml dH2O
    300 µl
    NADNa (100 mM)
    		dissolve 0.02 g NADNa in 300 µl dH2O
    4 x 60 µl
    dNTPs (100 mM, each)
    up to 6 ml
    dH2O

    aliquot á 350 µl