You want to amplify the vaccinia virus a52 gene by pcr...

You want to amplify the vaccinia virus A52 gene by PCR (polymerase chain reaction) and insert it into the pGEX vector. The 5\deg - and 3^(') - ends of the A.52 sequence ace given below, as well as a map of the pGEX recter a) Design appeopriate PCR primers, provide their sequesses, und levefly explain your coesiderations. c) Explain what the successfully made pGEX-AS2 exprevion vector can be used for Yaccinia vinus A 52 meacore: 5"-mpenemplemprtys-

Transcript

00:05 So you're trying to clone a novel protein called g1 into a vector.

00:14 So now what you have is the viral dna sequence of protein g1.

00:18 And what you're going to do is that you want to put to a restriction site in the beginning and end of this g1 dna sequence.

00:27 And of course, there also will be an n -terminal tag, which is hexahistidate.

00:32 This is going to be before the bamatu1 site.

00:35 So now you want to design two pcr primers that you would eventually use to clone into the vector.

00:43 So according to the figure, you should put bamage 1 in the beginning of the sequence and the zoh1 site at the end of the sequence.

00:51 So bamich 1 would be at 5 priman and zohan is going to be on a 3 primate.

00:57 And you should start out with the restriction sequence and then you will follow by 18 nucleotides.

01:06 That is complementary to the coding region of the g1.

01:10 All right, so now i have already copied the sequence to the board.

01:15 I did not copy every single nucleotide because you only need the beginning and end 18 nucleotides to design a primer.

01:21 The middle sequence is, i represent it, use a box because you do not need it.

01:28 All right, so the top strand is the coding strand of g1, and the bottom is template strand.

01:33 So according to our figures, bamage 1 should be at the 5 prime end.

01:39 So the bamich 1 i'm going to use red is going to be somewhere here.

01:43 This is little tail is bamish 1.

01:45 So this is the 5 primate.

01:48 And the bamich 1 should be followed by 18 nucleotides that is complementary to the bottom strand.

01:56 And so it's going to be at the 3 primate.

01:58 So you can see that i put an arrow there.

02:01 This tells you this is the forward primer.

02:03 The forward primer is going to be complementary to the three prime end of the bottom strip.

02:12 So you can see, this is what we are aiming for.

02:15 And the forward primer is going to go from five on the left and all the way to the left, or to the right to cover the bottom strip.

02:25 Now, this is the forward primer red one.

02:27 So remember, it's going to be complementary to the three prime end of the bottom strip.

02:33 Now what about zoh 1? zoh 1 is going to be at the 3 prime end of the coating string.

02:40 So i'm going to use green color...

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