txs at po.cwru.edu
Wed May 9 13:32:37 EST 2001
I agree with Emir. If your PCR product is less than about
700-800 bases and you have enough of it available you could
sequence it without cloning. To elaborate, if I wish to
sequence directly from the PCR product I dispense 100-150 ng
of purified PCR product, in a volume of about 10 ul, into
one tube and add 2 ul (5-20 pmol) of my forward primer to
it. I dispense the same amount of the same PCR product into
a second tube and add 2 ul of my reverse primer to it. Then
I have both preparations sequenced. The sequences you'll
get back should span the entire length of your PCR product.
Just remember that the sequence that had the reverse primer
in it will be the reverse complement of your PCR product. I
use a program called Chromas to help me analyze these
sequences to get a picture of the whole product. You can
check this program out at
http://www.technelysium.com.au/chromas.html (once again, no
affiliation). It's available as a download for a small fee
(about $75 US I think), and I think they allow you to
evaluate the program before you buy, so you might try it.
Emir Khatipov wrote:
> To sequence a PCR product you will need 2 primers (forward and reverse).
> Another thing to keep in mind is that the resulting readable sequence will
> lack first 10-20 bps, because they are 'fuzzy" due to a number of indigenous
> reasons of the sequencing procedure I do not really understand myself
> (someone enlighten please). Thus, the only sequence of that primary region
> will only be derived from the complement sequence. However, in most cases
> you will be able to reliably read 500-600 bps from one reaction. That means
> that if your gene is longer than that, you wouldn't be able to get the whole
> sequence. One of the solutions for this problem is to PCR amplify not just
> only the gene of your interest, but to take 20-30 bps extra bps from both
> You will avoid the above problems if you clone your gene into a vector.
> First, many of routinely used cloning vectors (like those TOPO vectors
> mentioned in one of the previous messages) have common promoter regions to
> which standard primers (T7, SP6, T3, ...) are available commercially, and
> sequencing firms usually have those and would not charge you for them. These
> promoters are usually located a few dozen bps upstream of the start of the
> insert sequence, so you will not have the problem of unreadable first 10-20
> ""Deanne Bell"" <dbell at qnis.net> wrote in message
> news:200105091531.f49FVvZ79883 at ns.qnis.net...
> > Thanks to everyone for the sequencing references!!
> > Now I have rookie question:
> > I understand that sequencing can be done directly from a PCR product
> > without cloning.
> > Under what conditions should cloning be performed?
> > I am now wondering if I really need to perform this step.
> > Deanne Bell
> > Molecular Markers Lab Technician
> > USDA Agricultural Research Service
> > 2021 S. Peach Ave.
> > Fresno, CA 93727
> > e-mail: dbell at qnis.net
> > phone: 559 453-3170
> > fax: 559 453-3088
> > ---
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