16. Suppose you are using a plasmid cloning vector that has no EcoRI sites ( $5^{\prime} G^{\wedge}$ AATTC $3^{\prime}$ ) in its polylinker because the particular drug resistance gene your vector contains has an EcoRI site within it.
a. How could you use the following two oligonucleotides (and DNA ligase) to ligate an insert that is an EcoRI fragment into the BamHI site ( $5^{\prime} G^{\wedge}$ GATCC $\left.^{\prime}\right)$ in the polylinker of your vector?
5' GATCCGGGGGGGGGG 3'
5' AATTCCCCCCCCCCG 3'

Question

Accepted Answer

∻Answer∻ ⚹ To ligate an insert that is an EcoRI fragment into the BamHI site of your vector using the given oligonucleotides and DNA ligase, follow these steps: ⚹ ∻Solution∻ ‖ a ⋮ Design complementary overhangs: The oligonucleotides provided have sequences that can create complementary overhangs for both EcoRI and BamHI sites. The first oligonucleotide (5' GATCCGGGGGGGGGG 3') has a BamHI overhang (5' GATC 3'), and the second oligonucleotide (5' AATTCCCCCCCCCCG 3') has an EcoRI overhang (5' AATT 3'). ‖‖ b ⋮ Anneal oligonucleotides: Mix the two oligonucleotides together and heat them to denature, then slowly cool to allow them to anneal, forming a double-stranded DNA with BamHI and EcoRI overhangs. ‖‖ c ⋮ Ligate insert: Use DNA ligase to ligate the annealed oligonucleotides into the BamHI site of the vector. The BamHI overhang of the vector will match the BamHI overhang of the oligonucleotide, allowing for successful ligation. ‖ ∻Key Concept∻ ⚹ Complementary overhangs ⚹ ∻Explanation∻ ⚹ The key to this process is designing oligonucleotides with complementary overhangs that match the restriction sites of the vector and the insert. This allows for precise ligation using DNA ligase. ⚹◊How can oligonucleotides and DNA ligase be used to ligate an EcoRI fragment into the BamHI site in a plasmid cloning vector?⍭ Generate me a similar question◊

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