It is often desirable to insert cDNAs into a cloning vector in su...

Question

It is often desirable to insert cDNAs into a cloning vector in such a way that all the cDNA clones will have the same orientation with respect to the sequences of the plasmid. This is referred to as directional cloning. Outline how you would directionally clone a cDNA library in the plasmid vector pUC18.

Accepted Answer

Welcome back, everyone. Here's our next question. What is the term used for cloning C DNA library in a plasmid vector in a way that ensures all the C DNA clones have the same orientation with respect to the plasmid sequences. Choice. A bidirectional cloning B, unidirectional cloning C directional cloning or D reverse cloning. So when we're making AC DNA library, those C DNA s are complimentary, that's where the sea comes from. Two M R N A s from an organism. So you're talking about a library essentially looking at expressed genes based on the M R N A s. So you don't have enrons or non coding regions and you're using restriction enzymes. So I'll put a to cut into fragments which are then cloned into plasmids. However, if your purpose is to have a library, you want to make sure that all of these fragments are going in the same order, so you can look at them and not get confused by things running in reverse. So if you want them to be in the same orientation that can be accomplished by using two different R E S or restriction enzymes to cut the two ends of the C DNA and then cut the plasmid with the same two enzymes. So then you have these sticky ends at the ends of your C DNA and they will only ligate with the correct sticky ends on the plasmid. So you can guarantee, always inserting into the plasmid in the same orientation with regards to the plasmid genes. And the term for that cloning it always in the same direction is choice C directional cloning. So that's the answer we're looking for. Choice C in this question. See you in the next video.

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