Designing PCR primers-How to check them?
brett
>> I have designed a set of primers for amplification of a 300 bp ds DNA
>> fragment from human genomic DNA.
>
>> I am interested to know how other people go about checking primers
>> which they have designed before actually ordering them.
>>
>> You can reach me at bk...@musicb.mcgill.ca or post a note here.
>
>> Thanx.
>>
>
I agree you should yield to your empirical results. However, you can
design your primers intelligently. One thing I've found useful is the
Amplify program available from the Indiana biosci ftp site. Briefly, you
can run the reaction "in silico", and it will point out annoyances such as
potential false priming sites in the template (assuming you know the
genomic sequence in your case), as well as primer-dimers, etc.
Brett Lindenbach
Lucille P. Markey Student in Human Pathobiology
Program in Immunology
Washington University - St Louis
br...@borcim.wustl.edu
Peter ---
fragment from human genomic DNA.
I would like to check these primers for any complementarity they may
have to repetitive sequences found in the human genome. I am
concerned that the presence of such complemantarity would result in
non-specific amplification.
Is there a database I can access to perform this search? If yes, how?
Graham Dellaire
> I have designed a set of primers for amplification of a 300 bp ds DNA
> fragment from human genomic DNA.
> I am interested to know how other people go about checking primers
> which they have designed before actually ordering them.
>
> You can reach me at bk...@musicb.mcgill.ca or post a note here.
> Thanx.
>
Unfortunately the only way you will really find out is by using them first and seeing how many bands
you get.
What is it for? ... sometimes more than one band is good (as in VNTR analysis) if you are typing
DNA or looking for markers.
GraHAM
_______________________________________________________________________
Graham Dellaire Snail Mail:
Red Cross, Research
McGill University Montreal Blood Services
Faculty of Medicine 3131 Sherbrooke St. East
Div. of Experimental Medicine Montreal, QC, Canada
E-mail: popa...@po-box.mcgill.ca H1W 1B2
B2...@musicb.mcgill.ca
WWW Page: http://www.medcor.mcgill.ca/EXPMED/expmed.html
Fax: (514) 525 0881
Voice: (514) 527 1501 ext 175
_______________________________________________________________________
Grant Morahan
bk...@musicb.mcgill.ca wrote:
> I have designed a set of primers for amplification of a 300 bp ds DNA
> fragment from human genomic DNA.
>
> I would like to check these primers for any complementarity they may
> have to repetitive sequences found in the human genome. I am
> concerned that the presence of such complemantarity would result in
> non-specific amplification.
>
> Is there a database I can access to perform this search? If yes, how?
Try the PRIMER program from the Whitehead - it will screen oligos for a
number of criteria, including checking vs a repeat seq database. Once you
get primers suggested by PRIMER, check them out for additional potential
problems using AMPLIFY, as recommended in an earlier reply to your post.
Good luck!
Regards, Grant
--
Grant Morahan, Ph.D.
The Walter and Eliza Hall Institute of Medical Research
Parkville, Victoria
AUSTRALIA
Tracy Aquilla
The best way to check your primers is to use them in a PCR! If you're using
a computer program you should do that before synthesizing the oligos. Once
you have them you may as well just try them out.
tracy
SEHuang
(around 70 degree C, you may even use one temp step for anneal AND
elongation) i have recently performed more than ten different PCR's with
different primer pairs on genomic DNA without bothering about self
priming, primer dimer and non-specific annealing to genomic targets - all
primer pairs worked well at first try. Depending on the protocol
(especially when ampifying PURIFIED DNA) hot start may be necessary.
Good luck
S. Huang
Children's Hospital
Boston