Editing Ligation and enzyme-independent assembly methods (section)

== Complementary annealing mediated by exonuclease (CAME) ==

[[File:CAME cloning.png|RTENOTITLE]]

[[File:CAME from mixed-fragments.png|RTENOTITLE]]

Use 30~50 ng of the vector and 30~50 ng of insert for CAME cloning. CAME reactions were typically carried out in a total volume 10 UL with 1 unit of enzyme and its corresponding buffer (Supplemental Table S1).

Purify PCR products (to eliminate dNTPs when Pfu or T4 DNA polymerase is used to recess the DNA ends).

(1) for T4 DNA polymerase, recess the DNA using T4 DNA polymerase at '''room temperature for 3 min,''' heat inactivate the T4 DNA polymerase at 75 oC for 5 min and anneal the DNA at 50 oC for 20~30 min; (2) for λ exonuclease, recess the DNA using λ exonuclease at 37 oC for 5 min, heat inactivate the λ exonuclease at 75 oC for 5 min and anneal the DNA at 50 oC for 20~30 min;

(3) for Pfu DNA polymerase, recess and anneal the DNA using Pfu DNA polymerase for 30 min at 50 oC; or (4) in cases of cloning of an insert whose ends are flanked by undesired sequences, recess the DNA using λ exonuclease at 37 oC for 30 min, heat inactivate the λ exonuclease at 75 oC for 5 min and anneal the DNA at 50 oC for 20~30 min, followed by using T4 DNA polymerase at 37 oC for 15 min to recess the 30 overhangs and fill-in gaps.

T4 DNA polymerase exhibits the highest exonuclease activity and that it can digest almost the entire 450 bp DNA fragment in 5 min.&nbsp;&nbsp;&nbsp;λ exonuclease and Pfu DNA polymerase show intermediate and the lowest activity. CAME using T4 DNA polymerase generates up to four times more white colonies than using&nbsp;λ exonuclease and Pfu DNA polymerase.&nbsp;

Pful Buffer:&nbsp;: 20 mM Tris (pH 8.8), 10 mM KCl, 10 mM NH4)2SO4, 2 mM MgSO4, 0.1% TritonX-100

&nbsp;T4 DNA polymerase Buffer: 67 mM Tris (pH 8.8), 17 mM (NH4)2SO4, 6.7 mM MgCl2, 1 mM DTT.

λ Exonuclease Buffer:&nbsp;67 mM Glycine-KOH (pH 9.5), 2.5 mM MgCl2, 50 μg/ml BSA, 0.2 mM dNTPs.&nbsp;λ exonuclease prefers 5’ phosphorylated substrates, T4 polynucleotide kinase (T4 PNK) ishould be included&nbsp;to phosphorylate PCR products.

a single exonuclease such as T4 DNA polymerase and λ exonuclease is ineffective for cloning the target fragment&nbsp;when a mixture of many other DNA fragments are present. Hoever, sequential treatment with λ exonuclease and T4 DNA polymerase generated the highest number of colonies with correct insert.&nbsp;&nbsp;Complementary sequences longer than 20 bp are required for this cloning