00:01
So you receive 10 nanograms of plasma dna and what is the first step you need to do to generate a source of dna for future cloning experiment and what method would you use and why? so you only have 10 nanograms of plasma dna so you do not have a lot.
00:16
So the very first step you if you want to establish a source of dna so you have to make more so you have to amplify the existing dna so that you have more of them.
00:31
So a very good way to amplify or make more dna is to use pcr.
00:40
The chain polymerase reaction basically if you have a template dna you have to know the primer that would be complementary to the flanking region of the dna that you want to amplify and then you will actually use primer and dntp to make new dna strand that is complementary to the template strand and now you can see you start from one dna molecule and then you end up having two strands and you repeat this cycle so for many times you'll get a lot of dna they're all identical so this is what we call the pcr reaction.
01:31
So from here you can see that the very first step you want to generate the source of dna for future cloning is to do pcr reaction to amplify this dna so you have a lot more.
01:51
The second step what feature of the plasma must you know to carry this out? why is this information necessary? so the very important plasmid feature that you know is going to be the selection in this case the antibiotic resistance gene.
02:19
So why is this feature important? so usually when we have a plasmid idea is that we are going to put our this is a circular plasmid we're going to clone our dna interest gene of interest the red piece into the plasmid to make a recombinant plasmid.
02:39
So once you have a recombinant plasmid you're going to introduce that into a host equalized cell so you can see this is the genome and this is called transformation.
02:50
So once you put the recombinant plasmid into the equalized cell they'll actually grow and proliferate and you have you will have a lot of bacteria cells that actually have this plasmid.
03:08
But there's also a possibility that do you have certain host cell that doesn't uptake the plasmid as you can see the bottom one doesn't have this plasmid.
03:19
So obviously we want to have a way to distinguish the two different kind of host.
03:26
The top one is being transformed with plasmid the bottom is just a regular e.
03:32
Coli without a plasmid.
03:34
So one way to distinguish these two is by using antibiotics resistant gene.
03:38
Let's say this recombinant plasmid has a ampicillin resistant gene.
03:43
Ampicillin is a common antibiotics so you can see that because the plasmid goes into the e.
03:52
Coli and ampicillin resistant gene is being expressed.
03:58
So the top host cell will become ampicillin resistant because the plasmid expressed the gene product.
04:10
So then the top one is resistant.
04:13
The bottom e.
04:15
Coli does not have the plasmid so it is sensitive to ampicillin treatment.
04:19
So this is going to be ampicillin sensitive.
04:22
So we're going to have a medium that actually supplied with ampicillin.
04:27
So the ampicillin in the medium is going to kill the bottom one that doesn't have a plasmid...