DESIGN AN EXPERIMENT FROM THE DETAILS BELOW You need to perform an experiment th
ID: 58099 • Letter: D
Question
DESIGN AN EXPERIMENT FROM THE DETAILS BELOW
You need to perform an experiment that will result in the cloning of a gene into the
expression vector pUC18.
A map of pUC18 with all features indicated, including the multi-cloning site, may be easily
obtained by typing the vector name into Google.
The insert containing the gene is a blunt-ended fragment that was not generated with a
restriction enzyme. The name and size of the gene is irrelevant. We are interested only in the
cloning strategy. However, the insert is 2.4 kb, a map of which is shown below with unique
restriction sites indicated.
In the scheme that you devise, be sure to include any controls; and a simple experimental
way of determining that the gene is in the correct orientation (you cannot for instance simply
send it out to be sequenced).
Explanation / Answer
Some of the information furnished here are ambiguous. But let me interpret all possible alternatives.
It was mentioned that the gene is a part of the larger 2.4 kb insert (and the sequence is not at all irrelevant in this case). As you are going to express it into pUC18, thyen I assume that the sequence is known to you. Design primers and append restriction sites to them of your favourite restriction enzymes, based on the pUC18 cloning site. Amplify it, digest both the amplicon and the vector, then ligate. Transform to competent E. coli along with the intact pUC18 as a control.
If the sequence is unknown, you only have to go for blunt end ligation and one more extra cloning step. Blunt end cloning is notoriously difficult. the CloneJet PCR-cloning kit (Thermo) can be used and the best method for this purpose. Get the clone, amplify with primers designed for the flanking regions of the insert ends (vector ends) with pUC18 restriction site appended, clone into pUC18.
Hope this helps.
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