Question

Please help to chose Sample and Provide a reason why you chose it.

Question 9 After 2 hours, a DNA gel is run on 4 different ligation samples. Each sample is run twice, once after incubating in Xhol and again after incubating in Ndel AND Xhol. Please identify which sample correctly incorporated the DNA into the vector. Note PET-28a has an approximate weight of 5400 bp. Why did you pick this sample? Type your response here 121 TITULLIITTI

Answer 1

Given the approximate size of pET 28a vector = 5400 bp or 5.4 Kb.

We are digesting four different samples, where a DNA fragment has been cloned in pET28a. The use of two enzymes Xho 1 and Nde 1 indicate directional cloning of the DNA fragment, as these two regions are found in the MCS of the cloning vector. This means that digestion with a single enzyme, Xho 1 , should linearlize the plasmid, and digestion with Xho 1 and Nde 1 should release the cloned fragment.

If we look at sample 1, digestion with single as well as double enzyme has resulted in the release of fragment of ~1.3 Kb. so this is not the right sample, unless a single enzyme is used for cloning. but this is not the case here.

Sample 2: This shows a linerarized vector at ~10 Kb, which is doube the size of pET 28a, but digestion with two enzymes resulted in a ~5 kb and a ~1.3 kb fragment, whcih does not add up to the ~10kb vector. This is not the right sample either.

Sample 3: This appears to the the right sample. Digestion with Xho 1 shows a linearized vector at ~6.3 kb and with both enzymes there are two bands , one at ~5 kb and another at ~1.3 kb. This does add up to the original size of the linearized vector when xho 1 alone was used.

Sample 4: This appears wrong, as digestion with xho 1 yields a different size fragment of ~2 kb along with the ~5kb fragment. The total size here is ~7 kb. whereas digestion with both enzymes yielded ~5 kb and ~1.3 kb fragment. The same vector cannot be of two sizes 7 kb and 6.3 kb!

The sample that correctly incorporated the DNA into the vector therefore, is sample 3.