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Team:Newcastle/Gibson Cloning
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| - | + | =Gibson Cloning= | |
| - | + | ==Background== | |
| - | This is a one-step isothermal method for assembling overlapping DNA fragments. Please see ''[http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html Enzymatic assembly of DNA molecules up to several hundred kilobases]'', Gibson et al. (2009). | + | This is a one-step isothermal reaction method for assembling overlapping DNA fragments. Please see ''[http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html Enzymatic assembly of DNA molecules up to several hundred kilobases]'', Gibson et al. (2009). |
| - | + | ==Reaction buffer recipes== | |
| - | + | ===5X isothermal reaction buffer=== | |
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|H<sub>2</sub>O | |H<sub>2</sub>O | ||
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| - | | | + | |Make up to 3 ml final volume |
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| - | + | ===1.33X Master Mix=== | |
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This makes 25 aliquots of 15 ul each. Store at -20°C. Although the mixture can be freeze-thawed and is stable for a year, we generally plan experiments to use an entire aliquot so they never go through the freeze-thaw cycle. The 1.33X Master Mix is in this case based on the volume of the Taq ligase from the manufacturer. We make the mix straight into the Taq ligase tube because it is important to work quickly with everything on ice. The T5 exonuclease may be difficult to accurately measure out and the water volume is great enough to allow for a 10X dilution so 20 ul of the dilution can be added to the mix. | This makes 25 aliquots of 15 ul each. Store at -20°C. Although the mixture can be freeze-thawed and is stable for a year, we generally plan experiments to use an entire aliquot so they never go through the freeze-thaw cycle. The 1.33X Master Mix is in this case based on the volume of the Taq ligase from the manufacturer. We make the mix straight into the Taq ligase tube because it is important to work quickly with everything on ice. The T5 exonuclease may be difficult to accurately measure out and the water volume is great enough to allow for a 10X dilution so 20 ul of the dilution can be added to the mix. | ||
| - | + | ==PCR product purification== | |
| - | PCR products that | + | PCR products that come from a plasmid template need to be purified on a 1% agarose TAE gel (adjust the gel and well size to accommodate the entire PCR (Phusion) reaction. This procedure is generally a good idea to reduce unwanted assemblies from minor aberrant PCR products. The Qiagen MiniElute gel extraction kits have been successful for this purpose as the final volume/concentration is approximately right for the assembly protocol. |
| - | + | ==One-step isothermal assembly== | |
For ~6 kb fragments, use 10-100 ng DNA of each DNA in 5 ul final volume. Scale accordingly for fragment sizes (molar ratios). | For ~6 kb fragments, use 10-100 ng DNA of each DNA in 5 ul final volume. Scale accordingly for fragment sizes (molar ratios). | ||
# On ice, add 5 ul DNA to 15 ul thawed 1.33X Master Mix | # On ice, add 5 ul DNA to 15 ul thawed 1.33X Master Mix | ||
# Incubate at 50°C for 60 minutes | # Incubate at 50°C for 60 minutes | ||
| + | |||
| + | N.B.: The amount of T5 exonuclease varies with the size of the overlap. Current amount is for ~40bp. | ||
Latest revision as of 12:46, 26 October 2010
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Contents |
Gibson Cloning
Background
This is a one-step isothermal reaction method for assembling overlapping DNA fragments. Please see [http://www.nature.com/nmeth/journal/v6/n5/abs/nmeth.1318.html Enzymatic assembly of DNA molecules up to several hundred kilobases], Gibson et al. (2009).
Reaction buffer recipes
5X isothermal reaction buffer
| 5X final | Amount | Stock concentration |
|---|---|---|
| 25% PEG-8000 | 0.75 g | powder |
| 500 mM Tris-HCl pH 7.5 | 1.5 ml | 1M |
| 50 mM MgCl2 | 75 ul | 2 M |
| 50 mM DTT | 150 ul | 1 M |
| 1 mM dATP | 30 ul | 100 mM |
| 1 mM dTTP | 30 ul | 100 mM |
| 1 mM dCTP | 30 ul | 100 mM |
| 1 mM dGTP | 30 ul | 100 mM |
| 5 mM NAD | 300 ul | 50 mM |
| H2O | Make up to 3 ml final volume |
1.33X Master Mix
| 1.33X | Amount |
|---|---|
| 5X isothermal buffer | 100 ul |
| T5 exonuclease 1.0 U/ul | 2 ul |
| Phusion DNA pol 2 U/ul | 6.25 ul |
| Taq DNA ligase 40 U/ul | 50 ul |
| H2O | 216.75 ul (375 ul final volume) |
This makes 25 aliquots of 15 ul each. Store at -20°C. Although the mixture can be freeze-thawed and is stable for a year, we generally plan experiments to use an entire aliquot so they never go through the freeze-thaw cycle. The 1.33X Master Mix is in this case based on the volume of the Taq ligase from the manufacturer. We make the mix straight into the Taq ligase tube because it is important to work quickly with everything on ice. The T5 exonuclease may be difficult to accurately measure out and the water volume is great enough to allow for a 10X dilution so 20 ul of the dilution can be added to the mix.
PCR product purification
PCR products that come from a plasmid template need to be purified on a 1% agarose TAE gel (adjust the gel and well size to accommodate the entire PCR (Phusion) reaction. This procedure is generally a good idea to reduce unwanted assemblies from minor aberrant PCR products. The Qiagen MiniElute gel extraction kits have been successful for this purpose as the final volume/concentration is approximately right for the assembly protocol.
One-step isothermal assembly
For ~6 kb fragments, use 10-100 ng DNA of each DNA in 5 ul final volume. Scale accordingly for fragment sizes (molar ratios).
- On ice, add 5 ul DNA to 15 ul thawed 1.33X Master Mix
- Incubate at 50°C for 60 minutes
N.B.: The amount of T5 exonuclease varies with the size of the overlap. Current amount is for ~40bp.
Transformation of E. coli may be done with 5-20 ul of the assembly mix depending upon the cells and method of transformation.
Go back to our Protocol List
   
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