PCR and plasmid vectors

Nick Jacobsen nickjacobsen at yahoo.co.uk
Fri Oct 19 05:12:17 EST 2001


hi Patrick,


ill try and answer your two questions:-

> Was wondering if anyone could help me with a problem.  A couple of questions?
> 
> What is the advantage of using PCR to generate a probe for screening a cDNA
> library?


as opposed to what? are you talking about using PCR to generate a
radioactive probe or to generate a probe template? if youre talking
about the former its better to use a basic incorporation method which
uses random hexamers to prime a Klenow polymerisation with an alpha
labelled radioactive dNTP, you can use a plasmid or PCR product as
your template it doesnt matter. This method is very quick. If you want
to use PCR to generate a radioactive probe it will take longer and to
be honest its not worth it because the method ive described is great.
Amersham do a kit called Megaprime which works fine.



> Why would the product of PCR be cloned into a plasmid vector before being
> sequenced?


well you may want to clone the PCR product so that you can use plasmid
based primers to sequence it as opposed to the PCR product primers
themselves although you can directly sequence PCR products usually
without problem. however sometimes PCR products dont sequence very
well using the product based primers. You may also want to clone a PCR
product for downstream use eg you might clone it into a reporter
vector and just sequence in there. Ultimately if youre talking about
generating a PCR product to use to generate a probe you dont need to
clone it just sequence it directly and then apply the clean PCR
product to the Megaprime reaction.


let me know if you need anymore help

cheers

Nick




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