Team:Newcastle/Gibson Cloning

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Revision as of 12:18, 5 August 2010

Gibson DNA assembly/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

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

@@ Line 85: / Line 85: @@
 ===PCR product purification===
-PCR products that came 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.
+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===