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General primer design questions

General primer design questions - Protocols and Methods Forum

General primer design questions - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 08-31-2003, 12:15 PM
Peter Frank
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Default General primer design questions



1) Can anyone give me any references explaining the theory behind the
thermodynamic principals of primer design?

2) When designing primers for analytical purposes only (i.e. for
example by using RT-PCR to see whether a gene is expressed or not),
what is a "good" length for the amplified product? My guess is 100 to
200 bp are enough. Am I right? And again when designing primers for
analytical purposes only, are there any more or less suitable
locations for the primers? Should the primers be located rather at the
beginning, in the middle or the end of the sequence to be amplified?
Or doesn't it matter?

Peter
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  #2  
Old 09-01-2003, 02:29 PM
Peter Ashby
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Default General primer design questions

In article <[Only registered users see links. ]>,
Peter Frank <[Only registered users see links. ]> wrote:


Depends on your sequence and the sequence of any homologues. If two
genes are identical at the 5' end but diverge at the 3', if you design
your primers to the 5' end you will detect both, the 3' end only one. If
two sequences are close but not identical make sure you anchor the 3'
ends of your primers in the bases that vary to avoid overlap and keep
stringency high.

Peter

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Peter Ashby
School of Life Sciences, University of Dundee, Scotland
To assume that I speak for the University of Dundee is to be deluded.
Reverse the Spam and remove to email me.
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  #3  
Old 09-02-2003, 03:30 AM
Eric R. Hugo
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Default General primer design questions

Peter Ashby <p.r.ashby@MAPS.dundee.ac.uk> wrote in news.r.ashby-
[Only registered users see links. ]:


The size range you mention is good for both conventional and real-time PCR
(qPCR). For the primer sets I have designed in the past year (~50 pairs) I
have just a couple of simple rules I try to follow:
1. Try to have the primers span a fairly large intron so that genomic DNA
will not be amplified.
2. Design all your primers around the same target melting temp (+/- 1 C) I
generally shoot for 60 C.
3. Use reliable tools for the design work and analysis. The Integrated
DNA Technologies web site ([Only registered users see links. ] free tools available
online that work quite well. I always BLAST and check the generated
primers for secondary structure before I order them.

Out of the past thirty or so sets I've designed and made only one set was a
flop and only two others needed optimization from tmy standard conditions.
I hope this info is useful.
Eric
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  #4  
Old 09-02-2003, 01:17 PM
Ferdinando Pucci
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Default General primer design questions

On Tue, 02 Sep 2003 03:30:47 -0000
"Eric R. Hugo" <[Only registered users see links. ]> wrote:


May it be a good idea to try DNA-PNA-DNA chimeras to optimize
hybridation (PNA hybrids more stricly than DNA, a single mismatch
brokes the interaction) keeping the ability to be processed by Taq Pol
thanks to the two cap of DNA (long the minimum for the binding of Taq
plus 2-3 Nu, for restore the dsDNA structure)?


--
All laws are simulations of reality.
-- John C. Lilly
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