TITLE: Plasmid Modification by Annealed Oligo Cloning
AUTHORS: Addgene the nonprofit plasmid repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes plasmid modification by annealed oligo cloning. To see the full abstract and additional resources, please visit the </span><a href="https://www.addgene.org/protocols/annealed-oligo-cloning/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Addgene protocol page</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Designing overlapping oligos]
Briefly, we will design overlapping oligos that once annealed can be cloned directly into the overhangs generated by restriction digest of existing sites in the original vector.To add NdeI, PacI, AscI and MfeI sites between the EcoRI and SalI sites of the vector, we design a top oligo with each of the additional sites in tandem (NdeI - CATATG, PacI - TTAATTAA, AscI - GGCGCGCC, MfeI - CAATTG). The bottom oligo will be the reverse compliment so that they can anneal.We also need to include additional bases to complement the overhangs generated when digesting the vector with EcoRI and SalI (see diagram). To do this, we add 5' - AATTC and G - 3' to the top oligo and 3' - G and CAGCT - 5' to the bottom oligo, making our final oligos 34 bp each:Order the following oligos from your favorite oligo synthesis company:  AB1Top oligo:5' - AATTCCATATGTTAATTAAGGCGCGCCCAATTGG - 3'2Bottom oligo:5' - TCGACCAATTGGGCGCGCCTTAATTAACATATGG - 3'
Note, we could leave off the 3’ G on each oligo (and the complementary C of the other oligo), but this would destroy the EcoRI and SalI sites in the final vector.
AB1Top oligo:5' - AATTCCATATGTTAATTAAGGCGCGCCCAATTGG - 3'2Bottom oligo:5' - TCGACCAATTGGGCGCGCCTTAATTAACATATGG - 3'
Note, if you plan to phosphatase treat your cut vector it is necessary to use 5'-phosphorylated oligos. This is an option that can be added when ordering them or can be performed enzymatically later.
?. [Digest and Purify Vector]
While waiting for your oligos to arrive, conduct a restriction digest of  of vector with EcoRI and SalI
1 µg
?. [Digest and Purify Vector]
Run an agarose gel and cut out the band containing your vector DNA
?. [Digest and Purify Vector]
Gel purify your DNA away from the agarose using a commercially available kit or standard protocol.
?. [Anneal Oligos]
Anneal oligosThe oligos should be resuspended in annealing buffer (10mM Tris, pH7.5-8.0, 50mM NaCL, 1mM EDTA) and mixed in equimolar concentrations. We recommend mixing  each in a total volume of  - add additional annealing buffer if necessary to get to 50μL. Efficient annealing can be achieved by one of two methods:
2 µg
50 µl
?. [Anneal Oligos]
Method 1Place tube in - hot block and leave for -.Remove the hot block from the heat source (turn off or move block to bench top) allowing for slow cooling to  (~).
90 °C
95 °C
0 Room temperature
?. [Anneal Oligos]
Method 2Place tube in a thermocycler programmed to start at  for .Then, gradually cool to  over .
95 °C
25 °C
?. [Ligitation]
Dilute of annealed oligos with  nuclease-free water and quantify the concentration (should be about 8ng/μl).
5 µl
45 µl
?. [Ligitation]
Mix the annealed oligos with cut vector in molar ratios (vector:insert) between 4:3 and 1:6 in a standard ligation reaction (ex. to ligate an annealed oligo insert of 50bp in length into a 5kb vector, mix 100ng of the vector with - of annealed oligos).
750 µg
6 ng
?. [Ligitation]
Transform- into your favorite competent bacteria and plate.
2 µl
3 µl
?. [Ligitation]
Be sure to pick multiple colonies for mini-prepping and verify insert by sequencing.