AQA BIOL5 ~ 17th June 2013 ~ A2 Biology

I don't mean to sound rude but I'm having trouble understanding the menstrual cycle (lord knows why since I'm a girl myself) but this looks like a really good poster! Any chance you would be willing to post a view from the front that is readable? I understand if you don't want to advertise your work- just thought it'd be worth asking!

haha thanks yeah sure I'd be happy to if I can take a good enough picture only thing is, i can't do it until Monday night, sorry.

Can someone help me?
I'm having trouble understanding the Sanger sequencing method.

So, say you've got a single strand of DNA you want to sequence. You use restriction endonucleases to cut it up. And you have four test tubes, each with DNA primers, DNA polymerase and radioactively labelled terminators (each tube is for one specific base), right? But then this is where I get confused.

Apparently PCR is involved, but I don't know why. Does each test tube get exactly the same fragments of the DNA? Or does each test tube have different fragments? Where, if at all, does PCR come into it?

Anyone got any good clear notes on this?

I think PCR gets used to amplify the amount of the single stranded peices of DNA you want to sequence, so the gel eelctrophoresis pattern you get at the end is more accurate and detailed.

Oh, ok. Wait, are restriction endonucleases even actually used in the Sanger method? I'm so confused. Does each test tube get exactly the same strand of DNA?

In the sanger method you dont have restriction endonucleases, but if you have a large piece of DNA you want to sequence you'll have to use restriction endonucleases to cut it down, then sequence each fragment using the sanger method.

Yep, each test tube has the same strand of DNA.

In the sanger method you dont have restriction endonucleases, but if you have a large piece of DNA you want to sequence you'll have to use restriction endonucleases to cut it down, then sequence each fragment using the sanger method.

Yep, each test tube has the same strand of DNA.

Do we need to know about Southern blotting? Also, do we only need to know of gel electrophoresis for the Sanger method (DNA sequencing)?

Yes and Yes.

James A!

You are good at biology, will you help me?

Does each test tube have many copies (produced by PCR) of the original DNA strand? And so does each test tube need to have lots of primers to attach onto each copied DNA strand?

All my books make out like there's just one strand and one primer in each tube :\

Firstly, I'm not good at Biology, it's my worst academic subject !!

Secondly...

For PCR, you obviously start off with one DNA strand you wish to replicate in a test tube. Add your primers, free nucleotides and your taq dna polymerase to the test tube. The machine is computer controlled and it can vary the temperature automatically.

Remember the PCR goes through many cycles which in total takes hours to complete, but produces MANY copies of the existing strand, so obviously you need to have many primers otherwise the PCR won't work. You also need to include many free nucleotides..


Think about it carefully, if there was one primer, then the reaction would stop after the first cycle!!! You need many primers to keep the reaction going!

Oh, but I've seen you around a lot on these Biology threads And you always seem to know what you're talking about lol.

I meant for the Sanger Sequencing method! I think I get PCR, I'm just unsure where it comes into the Sequencing.

Could you tell me if you think this is right for the Sanger Sequencing?

1) There are four test tubes, one specified for radioactive terminators of one base (A,T,G or C).
2) The single DNA strand to be sequenced is multiplied using PCR.
3) Numerous copies of the strand are put in each test tube with DNA primers (to help DNA polymerase attach free nucleotides), free DNA nucleotides, DNA Polymerase and the terminators.
4) In each test tube, DNA Polymerase begins to build a chain complementary to the template strand using the free nucleotides.
5) A terminator will bind to the template, stopping the synthesis at that point.
6) This produces DNA fragments of different lengths in each test tube, which are then separated out using gel electrophoresis.

Correct!

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Did my first essay today, and i took well over 45 minutes to do the essay :| Any tips on how to manage time properly?

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