Team:UC Davis/Protein Engineering Gibson

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<p>Gibson Cloning Protocol<br>
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Adopted from:  Gibson et al.  Nature Methods v. 6 n. 5; 343 (2009)</p>
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<p>DNA NEEDED<br>
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1.  Linearized dsVector<br>
 +
*This can be generated through restriction digests or PCR amplification of the backbone<br>
 +
2.  Linear dsDNA Inserts (1-10 chunks)<br>
 +
*Each piece needs to have both a 5’ and 3’ overlap with the subsequent piece in the assembly<br>
 +
*The overlaps should have a Tm of 65-75 (generally 20-40 base pairs)<br>
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*If overlaps have polyG (>4) missanealing can occur and the reactions may fail</p>
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<br><br>
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<p>PREPARE STOCK SOLUTIONS:<br>
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1. Prepare 5X ISO buffer (25% PEG-8000, 500 mM Tris-HCl pH 7.5, 50 mM MgCl2, 50 mM DTT, 1 mM each of the 4 dNTPs, and 5 mM NAD). Six ml of this buffer can be prepared by combining the following:<br>
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3 ml of 1 M Tris-HCl pH 7.5<br>
 +
150 μl of 2 M MgCl2<br>
 +
60 μl of 100 mM dGTP<br>
 +
60 μl of 100 mM dATP<br>
 +
60 μl of 100 mM dTTP<br>
 +
60 μl of 100 mM dCTP<br>
 +
300 μl of 1 M DTT<br>
 +
1.5 g PEG-8000<br>
 +
300 μl of 100 mM NAD<br>
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Add water to 6 ml<br>
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Aliquot 100 μl and store at -20 °C</p>
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<br>
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<ol>
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<li><p>Prepare an assembly master mixture. This can be prepared by combining the following:
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320 μl 5X ISO buffer
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0.64 μl of 10 U/ μl T5 exo
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20 μl of 2 U/μl Phusion polymerase
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160 μl of 40 U/μl Taq ligase
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Add water to 1.2 ml</p></li>
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<li><p>Aliquot 8 μl and store at -20 °C. This assembly mixture can be stored at -20 °C for at least one year. The enzymes remain active following at least 10 freeze-thaw cycles.
 +
This is ideal for the assembly of DNA molecules with 20-150 bp overlaps. For DNA molecules overlapping by larger than 150 bp, prepare the assembly mixture by using 3.2 μl of 10 U/ μl T5 exo.</p></li>
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</ol>
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<br>
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<p>PERFORM REACTIONS:<br>
 +
1. Thaw a 8 μl assembly mixture aliquot and keep on ice until ready to be used.<br>
 +
2. Add 3 μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).<br>
 +
3. Incubate at 50 °C for 15 to 60 min (60 min is optimal).<br>
 +
4. If cloning is desired, electroporate 1 μl of the assembly reaction into 30 μl electrocompetent E. coli.</p>
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Revision as of 23:34, 17 October 2014

UC Davis iGEM 2014

Design

Design

Build

Build

Test

Test

Gibson Cloning Protocol
Adopted from: Gibson et al. Nature Methods v. 6 n. 5; 343 (2009)

DNA NEEDED
1. Linearized dsVector
*This can be generated through restriction digests or PCR amplification of the backbone
2. Linear dsDNA Inserts (1-10 chunks)
*Each piece needs to have both a 5’ and 3’ overlap with the subsequent piece in the assembly
*The overlaps should have a Tm of 65-75 (generally 20-40 base pairs)
*If overlaps have polyG (>4) missanealing can occur and the reactions may fail



PREPARE STOCK SOLUTIONS:
1. Prepare 5X ISO buffer (25% PEG-8000, 500 mM Tris-HCl pH 7.5, 50 mM MgCl2, 50 mM DTT, 1 mM each of the 4 dNTPs, and 5 mM NAD). Six ml of this buffer can be prepared by combining the following:
3 ml of 1 M Tris-HCl pH 7.5
150 μl of 2 M MgCl2
60 μl of 100 mM dGTP
60 μl of 100 mM dATP
60 μl of 100 mM dTTP
60 μl of 100 mM dCTP
300 μl of 1 M DTT
1.5 g PEG-8000
300 μl of 100 mM NAD
Add water to 6 ml
Aliquot 100 μl and store at -20 °C


  1. Prepare an assembly master mixture. This can be prepared by combining the following: 320 μl 5X ISO buffer 0.64 μl of 10 U/ μl T5 exo 20 μl of 2 U/μl Phusion polymerase 160 μl of 40 U/μl Taq ligase Add water to 1.2 ml

  2. Aliquot 8 μl and store at -20 °C. This assembly mixture can be stored at -20 °C for at least one year. The enzymes remain active following at least 10 freeze-thaw cycles. This is ideal for the assembly of DNA molecules with 20-150 bp overlaps. For DNA molecules overlapping by larger than 150 bp, prepare the assembly mixture by using 3.2 μl of 10 U/ μl T5 exo.


PERFORM REACTIONS:
1. Thaw a 8 μl assembly mixture aliquot and keep on ice until ready to be used.
2. Add 3 μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).
3. Incubate at 50 °C for 15 to 60 min (60 min is optimal).
4. If cloning is desired, electroporate 1 μl of the assembly reaction into 30 μl electrocompetent E. coli.

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