Sequencing or Gene Specific Primer

Wolfgang Schechinger wolfsc at ibms.sinica.edu.tw
Thu Sep 20 01:45:45 EST 2001


Dear Kunika, sorry for the delay. I was out for a long weekend and then we
had little taifun Nari visiting us. Needed some days to dig my friends' home
out of the mud after the floodings finally had gone away. Now that the net
also is back again, so let's start... ;-)

When designing primers only to be used on plasmids, you have a lot of
freedom, since there won't be a lot of sites where they may bind
accidentally. Just avoid areas containing longer repeats and palindromes,
especially in the first say 12 bases of a primer. I personally like to minimize
or exclude 1 nucleotide. However, you may spent maximum effort in
optimizing the primers against each other.

Then use them (depending on on your cycler) on around 10ng of template.
Some parameters to start with: For a fast cycler like the Techne ProGene,
25-30 cycles at 55 degC annealing worked fine for me. Now with a very old
and slow Perkin Elmer Machine, I get better results with a touch down
program (68 to 55 degC, lower temp every two cycles by 2 degC, then run
to a total of 25-30 cycles at 55). You might use Pfu or another proofreading
enzyme since you need error free copies for expression. I's also a good
idea to sequence the construct before starting with the expression
experiments (and transfect some minipreps into cells and check for the
protein's activity if possible. Using something cheap like PEI or CaPO4 is
sufficient).

http://www.premierbiosoft.com/netprimer/netprimer.html is a nice tool for
checking compatibility against each other. However, the loading time of this
tool is horrific; if you only have a modem, better don't use it. But basically,
most of the time the job may be done by 'eyeballing'.

To minimize the effort on your problem, I'd design the primers in a way that
they are completely inside your target gene. Have the binding part starting
at ATG and the STOP codon (unless you need to remove it for adding some
tags) and reach into the coding sequence say maybe 18 to 21 bases. Then
add what's necessary to clone the gene into your favourite expression
vector (like pCDNA3). If the vector doesn't provide a Kozak consensus
element (you might have a look Anne Kozak' papers on that) for high
translation efficiency, add something like gccgccacc (that sequence has
worked fine for me) right in front of the starting ATG, furthermore the
restriction sites you need for cloning and some bases extra for the
enzymes to bind well (check NEBs catalogue, somewhere in the back.)
Check also that your sequence is in frame if necessary and for accidental
stop codons if you add some expressed elements already present in the
vector.

You also might use these primers for sequencing (as long as you get a pure
PCR product).

If these two primers don't work with your 'unknown' gene/vector, you still
might try any primer that could bind like there are CMV, T3, T7, Sp6,
anything binding to pUC/bBR322 backbones, AmpR, lacZ and so on to
bring enlightment into that business. Just dig in your freezer.

You also simply could cut out a fragment (assuming it's your known gene),
subclone and sequence it then from a known vector. To find out more about
the riddle gene/vector. If not, try to use enzymes common for cloning sites
(EcoRI, EcoRV, XbaI, XhoI, HindIII, BamHI, SalI (=sal1), HpaI, ... ). NdeI
(catatg) cuts some (mainly) genes right at the start codon and also could
be useful.

HTH

Wolfgang

> Dear Dr. Wolfgang Schechinger,
>
> Thanks for the response. What I was really interested to know is that if I'm
> amplifying a gene from a cDNA clone (that is I know the flanking region of the
> gene of interest as well as the terminal sequence of the gene of interest.)
> where should I design the primers? I presume that I should design the primers
> for the terminal sequence b/c I will be putting it in expression vector after
> the strong promoter.  What do you say?
>
> Secondly If I amplify another gene from another donor vector whose sequence is
> unknown, neither I know its terminal nor the flanking sequence, just I suspect
> that its the same gene that I planned to amplify from the cDNA clone. In  this
> case where I don’t know the sequence where to design the primers, I am planning
> to use either the Universal primer or the T3 or T7 primer, so here comes my
> actual question, can I use the universal primers or T3 or T7 primers available
> in my lab instead of arbitarory designing a new primers?
>
> I would appreciate your view and suggestions for this.
>
> Regards
>
> Kunika
>

[tiss mezzage wahs broduceRd using TYPO GENERATOR zoffwer]
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Dr. Wolfgang Schechinger
Lab N233 (c/o Dr. Steve Roffler)
Institute of Biomedical Sciences
Academia Sinica
128 Yen-Chio Yuan Rd. Sec.2
Taipei 115
Taiwan R.O.C.
Tel +886-2-2789-9152
Fax +886-2-2782-9142
Mobile +886-925-136893
e mail wolfsc at ibms dot sinica dot edu dot tw
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