Team:Warsaw/SyntheticCloning/SyntheticCloning

From 2011.igem.org

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<h2>Step by step guide to synthetic cloning</h2>
<h2>Step by step guide to synthetic cloning</h2>
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<img src="https://static.igem.org/mediawiki/2011/1/1f/Comix.png" width="700" align="middle">
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<div class="note">Rationale behind the protocol</div>
<div class="note">Rationale behind the protocol</div>
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<li>7. After ligation some linear DNA may still remain, you can get rid of it by using recBCD exonuclease and Exonuclease I</li>
<li>7. After ligation some linear DNA may still remain, you can get rid of it by using recBCD exonuclease and Exonuclease I</li>
<li>Linear DNA is a perfect template for RCR amplification with phi 29 polymerase. It generates the long linear concatamers of the DNA from circular templates. </li>
<li>Linear DNA is a perfect template for RCR amplification with phi 29 polymerase. It generates the long linear concatamers of the DNA from circular templates. </li>
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<li>After 12 hours of amplification DNA can be used in downstream processing</li>
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<li>After 16 hours of amplification you have the DNA of your construct</li>
</ul>
</ul>
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An excelent protocol on how to perform cell-free cloning is available here: <a href="http://www.biotechniques.com/multimedia/archive/00051/BTN_A_000113155_O_51382a.pdf" target="_blank">Takahashi H, Yamamoto K, Ohtani T, Sugiyama S. <i>Cell-free cloning using multiply-primed rolling circle amplification with modified RNA primers.</i> Biotechniques. 2009 Jul.
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</div>
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bellow is our protocol
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<br>
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<div class="note">This is how we did it</div>
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Our protocol:
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<div>
<ul>
<ul>
<li>1. Digest insert and vector with Fermentas Fast enzymes</li>  
<li>1. Digest insert and vector with Fermentas Fast enzymes</li>  
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<ul>
<li>insert with XBAI and at the same time add CIAP(Digest 1: 3ul reaction buffer; 1restriction enzyme; 5 ul DNA; 20ul water and 1ul CIAP), deactivate enzymes at 65C for 20 min, then digest insert  with PSTI(simply add 1ul of the enzyme).</li>
<li>insert with XBAI and at the same time add CIAP(Digest 1: 3ul reaction buffer; 1restriction enzyme; 5 ul DNA; 20ul water and 1ul CIAP), deactivate enzymes at 65C for 20 min, then digest insert  with PSTI(simply add 1ul of the enzyme).</li>
<li>vector with PSTI and at the same time add CIAP(Digest 1: 3ul reaction buffer; 1restriction enzyme; 5 ul DNA; 20ul water and 1ul CIAP), deactivate enzymes at 80C 20 min, then digest insert with SPEI(simply add 1ul of the enzyme)</li>
<li>vector with PSTI and at the same time add CIAP(Digest 1: 3ul reaction buffer; 1restriction enzyme; 5 ul DNA; 20ul water and 1ul CIAP), deactivate enzymes at 80C 20 min, then digest insert with SPEI(simply add 1ul of the enzyme)</li>
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<li> Prepare Lithium-botare agarose gel (1L 20X LB gel buffer=>950 mL of dH2O, 8.392 g lithium hydroxide monohydrate; 36 g of boric acid; pH 8.2. Volume to 1 L). Prepare gel as with TE or TBE, but you can run it on much higher voltage
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</ul>
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we use 300-400V and gel is ready in 15 minutes. Theoretically maximum you can use is max. 100V/cm, but we never tried that much.</li>
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<li> Prepare Lithium-botare agarose gel.  Gels in LB buffer can be can run on much higher voltage
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we use 300-400V and gel is ready in 15 minutes. Theoretically maximum you can use is max. 100V/cm, but we never tried that much. <ul><li>(1L 20 times concentrated LB gel buffer =>950 mL of dH2O, 8.392 g lithium hydroxide monohydrate; 36 g of boric acid; pH  8.2. Volume to 1 L)</li></ul></li>
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<li>Run the DNA on the gel and extract insert and vector</li>
<li>Run the DNA on the gel and extract insert and vector</li>
<li>Ligate:7 ul H20; 5 ul insetr; 5 ul vector; 2 ul buffer; 0.5 ul Ligase incubate for 15 min at 25C</li>
<li>Ligate:7 ul H20; 5 ul insetr; 5 ul vector; 2 ul buffer; 0.5 ul Ligase incubate for 15 min at 25C</li>
<li>Perform exonuclease digestion (2,5 recBCD exonuclease buffer; 2ul ATP; 1ul recBCD (Epicentre) ; 1 exonuclease I (Fermentas); 1 ul ligation mixture; 17,5 h20. Inactivate the reaction at 80C for 20 min</li>
<li>Perform exonuclease digestion (2,5 recBCD exonuclease buffer; 2ul ATP; 1ul recBCD (Epicentre) ; 1 exonuclease I (Fermentas); 1 ul ligation mixture; 17,5 h20. Inactivate the reaction at 80C for 20 min</li>
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<li>3. Set up the annealing mix as described:</li>
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<ul>
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<li>1ul of the reaction mix after exonuclease treatment</li>
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<li>1ul Phi29 polymerase buffer</li>
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<li>1ul PTO(phosphothioate protected) random RNA hexamers - 400umol, you can get them from IBA-GO</li>
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<li>RNAse free water to 10ul</li>
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</ul>
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<li>4. Set up the annealing reaction:</li>
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<ul>
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<li>heat mix to 95C</li>
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<li>cool down to 95C, slowly 0.1C/s works</li>
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</ul>
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<li>5. to the annealing mix add :</li>
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<ul>
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<li>1ul 10umol DNTPs - it is best to use RNAse free</li>
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<li>1ul phi29 polymerase from Epicentre</li>
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<li>2ul phi 29 buffer</li>
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<li>1ul diluted Pyrophosphatase from Fermentas. Diluted 1:100 according to the manual</li>
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<li>RNAse free water to 30ul final volume </li>
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</ul>
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<li>6. Incubate at 30C for 16h</li>
</ul>
</ul>
 +
An excelent protocol on how to perform cell-free cloning is available here: <a href="http://www.biotechniques.com/multimedia/archive/00051/BTN_A_000113155_O_51382a.pdf" target="_blank">Takahashi H, Yamamoto K, Ohtani T, Sugiyama S. <i>Cell-free cloning using multiply-primed rolling circle amplification with modified RNA primers.</i> Biotechniques. 2009 Jul.</a>
</div>
</div>
<br>
<br>
<div class="note">Results of the cell-free cloning</div>
<div class="note">Results of the cell-free cloning</div>
<div>
<div>
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<img src="https://static.igem.org/mediawiki/2011/d/d6/OzywianieBrika1.png" alt="Angry face" />
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5ul of the RC reaction were digested with Eco and PST. Insert and vector clearly visible, There are no unspecific products visible.
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<img src="https://static.igem.org/mediawiki/2011/f/f7/EcoPstTrawRCR.png" alt="Angry face" />
</div>
</div>
<br>
<br>

Latest revision as of 01:58, 21 September 2011

Example Tabs

Step by step guide to synthetic cloning



Rationale behind the protocol
  • 1. Cut DNA with one of the enzyme you want to use in cloning
  • 2. Dephosphorylate 5' phosphate - this prevents DNA pieces from self-ligation
  • 3. Cut DNA with the other of the enzyme you want to use in cloning
  • 4. Run DNA on the gel
  • 5. Extract vector and insert from the gel.
  • 6. Ligate
  • 7. After ligation some linear DNA may still remain, you can get rid of it by using recBCD exonuclease and Exonuclease I
  • Linear DNA is a perfect template for RCR amplification with phi 29 polymerase. It generates the long linear concatamers of the DNA from circular templates.
  • After 16 hours of amplification you have the DNA of your construct

This is how we did it
Our protocol:
  • 1. Digest insert and vector with Fermentas Fast enzymes
    • insert with XBAI and at the same time add CIAP(Digest 1: 3ul reaction buffer; 1restriction enzyme; 5 ul DNA; 20ul water and 1ul CIAP), deactivate enzymes at 65C for 20 min, then digest insert with PSTI(simply add 1ul of the enzyme).
    • vector with PSTI and at the same time add CIAP(Digest 1: 3ul reaction buffer; 1restriction enzyme; 5 ul DNA; 20ul water and 1ul CIAP), deactivate enzymes at 80C 20 min, then digest insert with SPEI(simply add 1ul of the enzyme)
  • Prepare Lithium-botare agarose gel. Gels in LB buffer can be can run on much higher voltage we use 300-400V and gel is ready in 15 minutes. Theoretically maximum you can use is max. 100V/cm, but we never tried that much.
    • (1L 20 times concentrated LB gel buffer =>950 mL of dH2O, 8.392 g lithium hydroxide monohydrate; 36 g of boric acid; pH 8.2. Volume to 1 L)
  • Run the DNA on the gel and extract insert and vector
  • Ligate:7 ul H20; 5 ul insetr; 5 ul vector; 2 ul buffer; 0.5 ul Ligase incubate for 15 min at 25C
  • Perform exonuclease digestion (2,5 recBCD exonuclease buffer; 2ul ATP; 1ul recBCD (Epicentre) ; 1 exonuclease I (Fermentas); 1 ul ligation mixture; 17,5 h20. Inactivate the reaction at 80C for 20 min
  • 3. Set up the annealing mix as described:
    • 1ul of the reaction mix after exonuclease treatment
    • 1ul Phi29 polymerase buffer
    • 1ul PTO(phosphothioate protected) random RNA hexamers - 400umol, you can get them from IBA-GO
    • RNAse free water to 10ul
  • 4. Set up the annealing reaction:
    • heat mix to 95C
    • cool down to 95C, slowly 0.1C/s works
  • 5. to the annealing mix add :
    • 1ul 10umol DNTPs - it is best to use RNAse free
    • 1ul phi29 polymerase from Epicentre
    • 2ul phi 29 buffer
    • 1ul diluted Pyrophosphatase from Fermentas. Diluted 1:100 according to the manual
    • RNAse free water to 30ul final volume
  • 6. Incubate at 30C for 16h
An excelent protocol on how to perform cell-free cloning is available here: Takahashi H, Yamamoto K, Ohtani T, Sugiyama S. Cell-free cloning using multiply-primed rolling circle amplification with modified RNA primers. Biotechniques. 2009 Jul.

Results of the cell-free cloning
5ul of the RC reaction were digested with Eco and PST. Insert and vector clearly visible, There are no unspecific products visible. Angry face

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