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    (Original post by ViolinGirl)
    :eek:

    What is Asepsis?
    Asepsis is the prevention of contamination by unwanted microorganisms. It is important to use aspectic technique, as microorgs can cause:
    1) Contamination of product leading to a loss of product (+ profit in industry)
    2) The microoganisms compete with the cultured organisms and thus result in slower growth rates + product production
    3) They can destroy the cultured microorganisms

    At lab level:
    -Wear gloves, tie hair back.
    -Flame any necks of cultures which ensures rising air to prevent microorgs falling in
    -Sterilise any equipment such as wire loops by placing in a bunsen flame for a few seconds, kills off microorgs.
    -Also can use an autoclave which is a high pressure and temperature steriliser
    -Disinfect work surfaces and can work in fume cupboards etc

    At Industrial level:
    -Steam sterilise equipment, and fermenter. Fermenter can be made of stainless steel to ensure microbes + nutrient material doesnt stick to it
    -Disinfect whole areas
    -Ensure there are filters on any inlets/outlets

    Errmmm ok, seriously at a loss of stuff to ask now lol. Describr a standard growth curve?
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    (Original post by Falcon91)
    Asepsis
    At lab level:
    more asepsis stuff..
    maintain a difference in air pressure between inside and outside of the room so air continually moves OUT of the room taking unwanted microorganisms with it

    rather unusual point, but i like it
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    This is the one exam I do not have to worry about, only need an E to average an A. Of course I still need a mid A to get an A* but its nice to have the leeway.
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    (Original post by BiGBaDBoO)
    This is the one exam I do not have to worry about, only need an E to average an A. Of course I still need a mid A to get an A* but its nice to have the leeway.
    what were your grades? for last year and this year and ISAs?........trying to work out what i need....
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    finally time to devote my time between biology and chemistry
    maths exams where a complete fail -.-, was better of spending my time revising bio or chem
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    hii chuck!! glad to see you're back and im sorry about your maths exam..
    emm have you finished the F215 revision cards by any chance?
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    could someone please clarify whether this is correct for sequencing a genome:

    - DNA is extracted and sheared into smaller sections
    - Multiple copies of one section of DNA are made using PCR
    primers are added
    DNA polymerase is added
    free nucleotides
    but also modified nucleotides with a fluorescent marker
    - Every time DNA polymerse lines up a modified nucleotide against template strand it is 'thrown off' stopping nucleotide chain from completing
    - This results in different chains all different lengths depending on when the modified nucleotide is added
    - Mixture of lengths of DNA are then separated by electrophoresis - shorter fragments move further across capillary tube
    - computer record colours as they pass tube e.g. 1st colour represents base at the end of the shortest chain
    - If there ae enough fragments every base is represented by a colour (sequence of colour = sequence of bases)
    - Each section of DNA is sequenced like this and then a computer programmer works out overall genome sequence


    Every book describes how to do this in different ways (like the CGP guide and the BAC's) and I'm not sure which way to learn it. Help? =(
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    (Original post by BHAM!)
    could someone please clarify whether this is correct for sequencing a genome:

    - DNA is extracted and sheared into smaller sections
    - Multiple copies of one section of DNA are made using PCR
    primers are added
    DNA polymerase is added
    free nucleotides
    but also modified nucleotides with a fluorescent marker
    - Every time DNA polymerse lines up a modified nucleotide against template strand it is 'thrown off' stopping nucleotide chain from completing
    - This results in different chains all different lengths depending on when the modified nucleotide is added
    - Mixture of lengths of DNA are then separated by electrophoresis - shorter fragments move further across capillary tube
    - computer record colours as they pass tube e.g. 1st colour represents base at the end of the shortest chain
    - If there ae enough fragments every base is represented by a colour (sequence of colour = sequence of bases)
    - Each section of DNA is sequenced like this and then a computer programmer works out overall genome sequence


    Every book describes how to do this in different ways (like the CGP guide and the BAC's) and I'm not sure which way to learn it. Help? =(
    That isn't genome sequencing involving BACs because idk about fluorescent nucleotides being used in that process. Isn't that automated DNA sequencing? They're both genome sequencing, you need to know both.

    There are two ways to sequence a genome. One is using the BACs and cutting them with different restriction enzymes to produce fragments of different sizes. The other way uses loads of copies of the SAME DNA fragment, the same primer, and then fluorescent nucleotides.
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    (Original post by The TSR Star.)
    That isn't genome sequencing involving BACs because idk about fluorescent nucleotides being used in that process. Isn't that automated DNA sequencing? They're both genome sequencing, you need to know both.

    There are two ways to sequence a genome. One is using the BACs and cutting them with different restriction enzymes to produce fragments of different sizes. The other way uses loads of copies of the SAME DNA fragment, the same primer, and then fluorescent nucleotides.
    the chain termination method, or the latter thing you described (with flourescent nucleotides /ddNTPS) couldn't sequence an entire genome in one go! that's why you have to place the genome into BACS and then decode their sequence using CTM piece by piece - then stick it back together via overlapping segments- So genome sequencing is a hybrid of the two, as far as I'm aware, as opposed to there being 2 distinct methods.

    With genome sequencing in mind, the OCR Book says that (once restriction enzymes have been applied to cut DNA fragments from BACS) they are separated by gel electrophoresis (DNA for this is usually double-stranded, fair enough). they are then sequenced using CTM - which is essentially interrupted PCR - which means the DNA sequence has to be single stranded! how does this jump occur? i know in PCR this is accomplished by heating to 95degrees, but the genome sequencing stages in the OCR book doesn't mention any heating for CTM. any ideas?
    sorry for a very wordy question haha
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    (Original post by mikey_g)
    the chain termination method, or the latter thing you described (with flourescent nucleotides /ddNTPS) couldn't sequence an entire genome in one go! that's why you have to place the genome into BACS and then decode their sequence using CTM piece by piece - then stick it back together via overlapping segments- So genome sequencing is a hybrid of the two, as far as I'm aware, as opposed to there being 2 distinct methods.

    With genome sequencing in mind, the OCR Book says that (once restriction enzymes have been applied to cut DNA fragments from BACS) they are separated by gel electrophoresis (DNA for this is usually double-stranded, fair enough). they are then sequenced using CTM - which is essentially interrupted PCR - which means the DNA sequence has to be single stranded! how does this jump occur? i know in PCR this is accomplished by heating to 95degrees, but the genome sequencing stages in the OCR book doesn't mention any heating for CTM. any ideas?
    sorry for a very wordy question haha
    Well now I'm confused.
    If they're the same then why would you cut the BACs with different restriction enzymes and then separate them by size BUT with the other one you do not have fragments of different sizes? Surely they are two different methods for the same thing.
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    5 days to learn all of F215....I love my life. :|
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    (Original post by The TSR Star.)
    Well now I'm confused.
    If they're the same then why would you cut the BACs with different restriction enzymes and then separate them by size BUT with the other one you do not have fragments of different sizes? Surely they are two different methods for the same thing.

    i dont see them as the same methods, but rather as complementary methods in this context, but..

    when you say 'with the other one' are you referring to CTM?
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    (Original post by mikey_g)
    i dont see them as the same methods, but rather as complementary methods in this context, but..

    when you say 'with the other one' are you referring to CTM?
    I mean the one with fluorescent nucleotides. And in reference to your first comment......erm so now you're agreeing with me? They're two different methods leading to the same thing.
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    (Original post by The TSR Star.)
    I mean the one with fluorescent nucleotides. And in reference to your first comment......erm so now you're agreeing with me? They're two different methods leading to the same thing.
    yeah, the fluorescent nucleotides one. not quite, i still dont agree, but maybe someone else can put their input to clarify with this? i maintain you can't sequence an entire genome using the 'fluorescent nucleotide' method alone, as it only works with up to fragments 750 base pairs long:

    "the sequencing reaction can only operate on a length of DNA of about 750 base pairs"

    so you shear the genome into BACS etc etc, then sequence each BAC using the nucleotide method, then put each BAC sequence back together. so its a hybrid of both methods as opposed to two distinct methods.

    just to check we're on the same page, do you disagree with that?:P
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    (Original post by aiden1234)
    what were your grades? for last year and this year and ISAs?........trying to work out what i need....
    I cant remember off the top of my head the individual UMS (I had my exam results sheet at the time). However If I remember correctly I was above 90% in all but the coursework and January module last year, all were A's.

    Remember this is just a guestimation assuming that the grade boundaries remain static, which never happens.

    If you would like aid in calculations.

    15% = First Jan Unit
    15% = Second Jan Unit
    25% = First June Unit
    25% = Second June Unit
    10% = First CWK
    10% = Second CWK

    After requesting the paper back from Jan, I had lost roughly 10 marks yet my UMS was 88/90. This illustrates how grade boundaries are as random as Seals in exam papers.
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    (Original post by mikey_g)
    yeah, the fluorescent nucleotides one. not quite, i still dont agree, but maybe someone else can put their input to clarify with this? i maintain you can't sequence an entire genome using the 'fluorescent nucleotide' method alone, as it only works with up to fragments 750 base pairs long:

    "the sequencing reaction can only operate on a length of DNA of about 750 base pairs"

    so you shear the genome into BACS etc etc, then sequence each BAC using the nucleotide method, then put each BAC sequence back together. so its a hybrid of both methods as opposed to two distinct methods.

    just to check we're on the same page, do you disagree with that?:P
    OHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH!

    So you see in the purple book, relating the the BAC bit step 3 (each fragment is sequenced using an automated process). That automated process is in fact the fluorescent nucleotides bit. Yep?

    Well how does that sequence a genome? I understand the method now, but at the end, when we compare the overlapping regions do we get the sequence of the BAC or the genome?
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    (Original post by BiGBaDBoO)
    I cant remember off the top of my head the individual UMS (I had my exam results sheet at the time). However If I remember correctly I was above 90% in all but the coursework and January module last year, all were A's.

    Remember this is just a guestimation assuming that the grade boundaries remain static, which never happens.

    If you would like aid in calculations.

    15% = First Jan Unit
    15% = Second Jan Unit
    25% = First June Unit
    25% = Second June Unit
    10% = First CWK
    10% = Second CWK

    After requesting the paper back from Jan, I had lost roughly 10 marks yet my UMS was 88/90. This illustrates how grade boundaries are as random as Seals in exam papers.
    Yes! I had 47/60 and my UMS was 88/90. And then I had 47/60 for Chemistry and it came out as 79/90. It's crazy stuff :p:
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    (Original post by The TSR Star.)
    OHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH!

    So you see in the purple book, relating the the BAC bit step 3 (each fragment is sequenced using an automated process). That automated process is in fact the fluorescent nucleotides bit. Yep?

    Well how does that sequence a genome? I understand the method now, but at the end, when we compare the overlapping regions do we get the sequence of the BAC or the genome?
    yep!
    i think you end up with a BAC sequence, and the book leaves it there. to be honest i've just memorised the steps and dare not question it haha. although if you're interested, i think CGP expands on it further...

    "finally, the DNA fragments from all the BACS are put back together, by computers, to complete the entire genome"

    in an exam situation i'd use the magic terms 'by computers' and 'overlapping fragments' to waffle through any of that part, if it was asked
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    (Original post by mikey_g)
    yep!
    i think you end up with a BAC sequence, and the book leaves it there. to be honest i've just memorised the steps and dare not question it haha. although if you're interested, i think CGP expands on it further...

    "finally, the DNA fragments from all the BACS are put back together, by computers, to complete the entire genome"

    in an exam situation i'd use the magic terms 'by computers' and 'overlapping fragments' to waffle through any of that part, if it was asked
    Should a massive question on this come up (a whole 8/9 marker). You, mikey_g, may be the sole reason that I get an A*.

    thankyou. :yep:
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    haha nice one, that's alright!
    although i doubt very much they'll be setting these sorta memory test questions. its all about knowledge application these days


    (Original post by BHAM!)
    could someone please clarify whether this is correct for sequencing a genome:

    - DNA is extracted and sheared into smaller sections
    - Multiple copies of one section of DNA are made using PCR
    primers are added
    DNA polymerase is added
    free nucleotides
    but also modified nucleotides with a fluorescent marker
    - Every time DNA polymerse lines up a modified nucleotide against template strand it is 'thrown off' stopping nucleotide chain from completing
    - This results in different chains all different lengths depending on when the modified nucleotide is added
    - Mixture of lengths of DNA are then separated by electrophoresis - shorter fragments move further across capillary tube
    - computer record colours as they pass tube e.g. 1st colour represents base at the end of the shortest chain
    - If there ae enough fragments every base is represented by a colour (sequence of colour = sequence of bases)
    - Each section of DNA is sequenced like this and then a computer programmer works out overall genome sequence


    Every book describes how to do this in different ways (like the CGP guide and the BAC's) and I'm not sure which way to learn it. Help? =(

    so in response to youu, the stages are as follows


    SEQUENCING ENTIRE GENOMES

    • Genomes are first mapped to identify which part of the genome (e.g. which chromosome) they have come from - location of microsatellites used (repetitive runs of base sequences)
    • Genome sheared (mechanically broken) into smaller fragments - known as the shotgun approach
    • Fragments placed into BACS and transferred to E.coli bacteria. As cells grow in culture, clone libraries are produced (many copies of the genome sections)

    • To sequence a BAC, cells containing specific BACS are taken and cultured. DNA is extracted from the cells and various restriction enzymes cut DNA fragments into different sizes.
    • Fragments separated by size using agarose gel electrophoresis
    • Each fragment is sequenced using an automated process (Chain termination method / or 'the fluorescent nucleotide thing')
    • Computer programmes compare overlapping regions from cuts made by restriction enzymes, in order to reassemble entire BAC, and then BACS are assembled together, forming completed code.


    So you're more or less right, but don't say copies are made using PCR, because whilst CTM is based upon PCR but it's different
 
 
 
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