Turn on thread page Beta
    • Thread Starter
    Offline

    2
    ReputationRep:
    Could anyone briefly explain the differences between PCR (polymerase chain reaction), Real-time PCR, and Multiplex PCR?
    Offline

    0
    ReputationRep:
    I don't know anything about multiplex PCR, but real time PCR is when the PCR machine is hooked up to a computer so you can watch the reaction happen in 'real time', usually in the form of a graph. This means you can see whether there is any product/contamination etc without having to run a gel first.
    • Thread Starter
    Offline

    2
    ReputationRep:
    (Original post by meeeee)
    I don't know anything about multiplex PCR, but real time PCR is when the PCR machine is hooked up to a computer so you can watch the reaction happen in 'real time', usually in the form of a graph. This means you can see whether there is any product/contamination etc without having to run a gel first.
    Thanks for the reply . So what would you actually see on the computer then? Some kind of absorption spectrum of the sample?
    • Thread Starter
    Offline

    2
    ReputationRep:
    BTW, I know what multiplex PCR is now. It is where you are simultaneously amplfying two different target sequences, usually using species/strain specific primers. It would allow you to analyse DNA extracted from a food sample for example, and determine if the E.coli present are of a pathogenic or non-pathogenic strain.
    • Thread Starter
    Offline

    2
    ReputationRep:
    Also, if anyone knows what 'minipreps' is then rep is available. This is with regard to identifying recombinant plasmids that have had a PCR product ligated into them.
    Offline

    0
    ReputationRep:
    I don't know if this is standard procedure, but when I did it, an extra reagent was added to the PCR mix, syber green which is fluorescent. This binds to the DNA, and when the strands separate, the fluorescence is lost, and this is what the computer records, as well as the temperature the strands separate. The computer also reverses the fluorescence, so on the graph, it looks like there is an increase in fluorescence when the strands separate. The graph then shows peaks at the temperature where the strands separate. Different DNA has different temperatures it will separate, so you do need to know that before you start. Other things like primer dimers, any negative controls etc will peak at different temps, so its easy to tell which is which. Hope all that made sense!
    • Thread Starter
    Offline

    2
    ReputationRep:
    (Original post by meeeee)
    I don't know if this is standard procedure, but when I did it, an extra reagent was added to the PCR mix, syber green which is fluorescent. This binds to the DNA, and when the strands separate, the fluorescence is lost, and this is what the computer records, as well as the temperature the strands separate. The computer also reverses the fluorescence, so on the graph, it looks like there is an increase in fluorescence when the strands separate. The graph then shows peaks at the temperature where the strands separate. Different DNA has different temperatures it will separate, so you do need to know that before you start. Other things like primer dimers, any negative controls etc will peak at different temps, so its easy to tell which is which. Hope all that made sense!
    Yep, thanks that is interesting and makes a lot of sense from some of the things i've been reading.

    So just to confirm, the fluorescent dye is used to determine when the strands separate? I assume that when it dissociates from the DNA, it must in some way change conformation or something like that so it is no longer fluorescent? (since it would still be in the sample).

    Oh, and if you have the time, how are primer dimers detected? Does the dye also associate with those?

    Sorry for the questions lol!
    Offline

    0
    ReputationRep:
    Yes, the dye is used to tell when the stands separate and the temp at which that occurs, but I don't know exactly how it works! The dye does also bind to the primer dimers, and those will separate at a different temp than the target DNA will if there is product. So on the graph you'll see a peak at a lower temp than the one for target DNA. BTW, the graph produced is called a melt curve analysis.
    • Thread Starter
    Offline

    2
    ReputationRep:
    Excellent, thank you very much for your help! I shall give you some rep tomorrow!
    Offline

    0
    ReputationRep:
    ah minipreps. From what I understand (which is pretty narrow), they're just kits you can get for purifying plasmid DNA from bacteria in liquid culture, without using that chlorophenol method. Basically, you centrifuge your bacteria into a little pellet, add some buffers to lyse the cells and precipitate out the crap, then pass the remaining solution through these special columns containing a membrane that DNA will stick to. Get rid of the solution that passes through, and then elute the DNA from the membrane with another solution to get your plasmids for transfection etc.

    For identifying recombinant plasmids, what I did was perfom a series of test digests i.e. examine the expected plasmid sequence and look for restriction endonucleases that would cut only once in the plasmid, then choose two of these, one to cut at a site in the ligated section and the other to cut outside of it. The fragment lengths obtained from digests with these enzymes should then tell you whether the ligation was sucessful or not.
 
 
 
Reply
Submit reply
Turn on thread page Beta
Updated: April 13, 2006
Poll
Do you think parents should charge rent?

The Student Room, Get Revising and Marked by Teachers are trading names of The Student Room Group Ltd.

Register Number: 04666380 (England and Wales), VAT No. 806 8067 22 Registered Office: International House, Queens Road, Brighton, BN1 3XE

Write a reply...
Reply
Hide
Reputation gems: You get these gems as you gain rep from other members for making good contributions and giving helpful advice.