Fw: sequence services
wgallin at gpu.srv.ualberta.ca
Wed May 9 12:24:46 EST 2001
You have to perform the subcloning if the band you have amplified
originates from more than one template sequence; otherwise, you get a
mixed sequence that will be impossible to read. For example, if you
have amplified from a gene family that has divergent sequence between
the primer sites.
On the other hand, if you are reasonably sure that your amplicon is
unique, sequencing the whole PCR product is quite doable, avoids extra
steps, and has the added advantage that any mistakes in sequence
introduced into single DNA molecules during the amplification are
drowned out by the other amplified products that don't have the error.
Deanne Bell wrote:
> 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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