These are not the physics vectors.

Biology Level pending

A vector is 1 kb long. Cloning is done, and a 1 kb insert is ligated into the vector, for a total of 2 kb, theoretically if the ligation worked. The ligation was left overnight at 4 C 4^{\circ} C and in the morning, was electroporated into bacteria. Antibacterial selection was performed, and the antibacterial resistance gene is found in the vector. When the bacteria grew up, miniprep was done to obtain the plasmids.

We can check if the cloning was done correctly using restriction digestion, and then running the digestion products through an EtBr agarose gel.

Which combination of restriction enzymes uses the minimum amount of restriction enzymes but will tell us whether the cloning worked?

Assume that there are no dead restriction sites, and assume that you had the choice of the restriction sites to affix to the inserts, and assume that all the possible restriction sites you could have ever wanted in the vector could be there, if you want them to be there. Also assume that if I say 2 or 3 restriction enzymes, they are all different enzymes, for different restriction sites. One specific restriction enzyme works on only one specific restriction site.

2 restriction enzymes; one for each different restriction site flanking the insert. 1 restriction enzyme; its corresponding site is found on both ends of the insert. 3 restriction enzymes; 2 different restriction sites flanking the insert, and 1 site in the vector. 1 restriction enzyme, whose site is found in the insert. 1 restriction enzyme; its restriction site flanks the insert, and is found once in the vector. 1 restriction enzyme for one restriction site found in the vector.

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1 solution

Hobart Pao
Sep 6, 2015

If you only use a restriction enzyme that flanks both sides of the insert, since the insert is the same size as the vector, you will only see one band on the gel, which is the 1 kb band. You won't know whether part of that 1 kb is the insert and part of that 1 kb is the vector AND the ligation worked, or that you only have the vector (and maybe the insert???). Because as long as the vector is there, the bacteria will survive, since the antibiotic resistance gene is in the vector, which is non dependent on the ligation.

If the restriction enzyme flanks both sides of the insert and has a site in the vector, if your ligation worked, you will see 3 bands: 1 1kb band for the insert, and 2 <1 kb bands that sum to 1 kb for the digested vector. If ligation didn't work, then you would only see 2 <1 kb bands that sum to 1 kb, and possibly a very faint band for 1 kb if any insert was taken up but not ligated.

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