Thus the 5 arm must be inserted into plasmid first if

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Thus the 5’ arm must be inserted into plasmid first. If the 3’ arm were inserted into the plasmid first, the SwaI would cut up the 3’ arm when the 5’ arm was being inserted. The resulting fragments might ligate in the wrong place and/or in the incorrect orientation. 3. Which pair(s) of restriction sites (if any) can be ligated to each other? Only consider the restriction sites provided above. No two different restriction sites provided above could be ligated to each other. 4. Why was ApaI included into the SwaI polylinker? Hint: what will you get if you digest this plasmid with ApaI and ran the result on an agarose gel as opposed to any other restriction site? There is only one restriction site for every restriction enzyme. Thus one of the restriction sites within the SwaI polylinker must be the same as one of the sites originally included on the plasmid. I had chosen ApaI. In this way, when the plasmid is digested with ApaI, two bands will result on agarose gel. 5. What would you do to test whether the SwaI polylinker had been successfully inserted into the ploxPFlpneo plasmid? There are two ways to test whether the SwaI polylinker had been successfully inserted. One could digest the plasmid with SwaI and observe 2 bands (at about 5.2Kb and 0.05Kb). Because the 0.05Kb band is difficult to resolve on agarose gel with the 5.2Kb (the 0.05Kb band will likely
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