00:01
A goal to make a glow -in -the -dark fish.
00:04
Your first step is to isolate the gene for green fluorescent protein from jellyfish.
00:08
Now you want to use pcr to isolate or amplify the gfp protein, or gene, i'm sorry, the sequence will represent the desired region you want to amplify.
00:18
So the question asks you to write out the first 10 bases for each of these primers you will use to amplify the desired region.
00:25
So i did not copy the entire sequence because you only need 10 nucleotides.
00:30
So you need two primers.
00:32
One primer is called the reverse primer.
00:35
The reverse primer is complementary to the three primers of the top strand of the template.
00:40
So i'm going to use a red color to show the reverse primer.
00:45
So the reverse primer has a five primers on the right side, and then it will be complementary to the region right here.
00:54
So c pair up with g, a pair up with t, t pair up with a, and the first 10 nucleotides.
01:12
And then you can see that the three primer is right there, and the primer will actually extend the new sequence at the three primer from right to left direction.
01:25
So the reverse primer is going to amplify the top strand.
01:29
Then you'll also have a forward primer, which will be complementary to the bottom strand, which is the three primer.
01:37
So the four primers, five primer is on the left side.
01:40
And again, it follow base pair rule.
01:43
So it start with t, g, a, t, t, a, g, c, t, a.
01:59
Again, 10 nucleotides.
02:01
So this is the three primer.
02:02
Again, the new nucleotide will be added to the three primer of the four primers, extend from left to right direction, which will amplify the bottom strand.
02:12
So now you have two primers, reverse and forward primers to amplify the entire region, and each 10 nucleotides.
02:23
Now the second question says that here is the beginning of the gfp protein, or here is the beginning of the gfp gene.
02:30
Given that this gene is read from left to right, that's the keyword.
02:36
Write out the mrna that will be produced when the gene is transcribed.
02:39
Since it's being read from five to three, i only copied the top strand.
02:45
This top strand is the coding strand, which will give you the coding information.
02:51
Now the coding information will tell what sequence is mrna.
02:55
So basically, coding strand of the dna has the same sequence as the mrna.
03:02
However, since it's a dna, you have a t in the sequence, but the t will become a u in mrna because there is no thymine in mrna.
03:16
Instead, you have uracil instead.
03:18
So the mrna sequence will basically be same as the top strand of the dna.
03:24
Five, also on the left side, so the same orientation, g, a, a, u, u, c, a, u, g, g, c, g, a, u, g, a, a, a, a, g, u, u, u, c, g, u, g, a, a, 3.
03:59
So this is mrna.
04:03
So once you have the mrna, the question asks you to translate your mrna into amino acid sequence.
04:09
So very first thing you want to find out the star codon.
04:13
A star codon is the beginning of amino acid chain.
04:18
Basically, ribosome is searching for a star codon.
04:22
So this is where the amino acid chain or polypeptide begins.
04:26
And the star codon is aug.
04:29
So after scanning the sequence from five to three of mrna, i find out my first aug right here.
04:36
So then you read it every three nucleotide.
04:39
Gcg is the second, aug is the third.
04:42
Now, when you second time you see aug, it's not a star codon.
04:45
So the new strand doesn't start over again.
04:48
It basically is a regular methionine.
04:52
So the next one, a, a, g, u, u, u, c, g, u, and g, a, a.
04:57
So as you can see, this is just the beginning part of the gfp protein.
05:02
So you're not expecting to see a stop codon.
05:05
So you will have a star codon, which is aug.
05:08
So the star codon here is also a methionine because this is the first amino acid of all the polypeptide...