pcr prob

ChenHA via methods%40net.bio.net (by hzhen from freeuk.com)
Mon Feb 25 07:59:11 EST 2008


Aawara Chowdhury wrote:
> In <1203893436.24100.0 from proxy01.news.clara.net>,
>  ChenHA <hzhen from freeuk.com> wrote:
> 
>> How do you know how much primers the original poster added?  
> 
> The amount doesn't matter - see below.
> 
>> It is for 
>> the original poster to comfirm or deny, or check by runnning the gel (as 
>> I stated, by using the same amount), not for you to assert.  Whoever 
>> gives you the idea the 5 or 10 picomoles is what everyone uses?  I 
>> don't, and I have done perhaps thousands of successful PCR reactions. 
>> And they do fluoresces, how brightly of course depends on how much you add.
>>
>> As it happens, I see this kind of things quite often, from my own PCR.
> 
> Ethidium bromide binds single-stranded DNA extremely poorly, if at all.
> It fluoresces when bound to RNA, simply because most RNA molecules do
> fold-back upon themselves to form partial duplexes.  Long ssDNAs also
> fluoresce in the presence of ethidium for the same reason.
> 
> I find it highly improbably that your ss oligonucleotides snap back on
> themselves sufficiently to fluoresce with ethidium, in the absence of
> polymerization that makes them ds.
> 

Are you in the habit of teaching your grandma to suck eggs?  Perhaps I 
should have added when I said that I have seen it often before, that I 
have also checked with running the primers by itself on the gel, and saw 
a bright glowing band?  And I have even ran primers down a gel 
filtration column and see big peak of aggregated primers.  Can that the 
cause of the bright bands? I don't know because it is not something 
worth investigating further, and all I can say is that I have seen it 
and primers alone often give big bright bands.

And where did you read how long the primers are that the original poster 
used?

I have read the other reply by someone else suggesting primer dimer, and 
I am merely offering an alternative explanation which satisfactorily 
explain in most cases what I have seen myself, and offered a few other 
suggestions on how to proceed further (how to check and how to resolve a 
problem like this, and yes, I have seen cases where it might be primer 
dimer), and to correct the impression that anyone need to redesign the 
primer if the PCR gives the correct band (usually you just need to 
adjust a few parameters and the result can be very good).  If the 
original poster ran his primer on the gel and see no bright band, then 
he will conclude that the other poster's suggestion of primer dimer is 
correct and I am wrong, no problem.  What you have contributed is 
precisely nothing except to annoy me and showed your own stupidity.  Now 
go away.



> AC


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