Editing Confusion Cloning (section)

== Confusion reaction: ==

50–200 ng linear vector<br>appropriate amount of insert DNA in a 1:1 to 10:1 molar ratio of insert to vector<br>1 ul 10X Confusion buffer (500 mM Tris–HCl, pH 7.5 at 25 oC, 100 mM MgCl2, 10 mM ATP, 10 mM DTT)<br>1 ul Confusion extract<br>ddH2O to a total volume of 10 ul.

Incubate at 37 oC for 1 h.<br>Transform homemade Top10 competent cells<br>

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In-Fusion PCR Cloning: Clontech

Cold Fusion Cloning Kit: SBI

Fast Seamless Cloning Kit: Dogene

CloneEZ Kit: Genescript

GENEART Seamless Cloning and Assembly Kit: Invitrogen

SLIC uses the 3' -&gt;50 exo-nuclease activity of T4 DNA polymerase to generate ssDNA overhangs in insert and vector which are required for the fusion of vector and insert fragments by single strand annealing with or without the addition of<br>RecA.

Exonuclease III induced ligase-free directional subcloning uses 3' -&gt;5' exonuclease activity of Exonuclease III to generate ssDNA overhangs which facilitate cloning.

Ribocloning uses Rnase A to cleave at single rC or rU bases that were introduced by PCR into vector and insert and subsequent heating to generate ssDNA overhangs for cloning.

Enzyme-free cloning creates complementary ssDNA overhangs by PCR with tailed primer sets and post-PCR denaturation-hybridization reactions.

In-Fusion PCR Cloning promotes PCR cloning by the In-Fusion enzyme, a poxvirus DNA polymerase with 3' -&gt;5'exonuclease activity.<br><br>

[[Category:Protocols]]