Sequencing

Byung-Hoon Kim byung-hoon.kim at uni-tuebingen.de
Wed Nov 17 05:22:16 EST 1999



"Hiranya S. Roychowdhury" schrieb:

> At 04:10 PM 11/16/99 +0100, Byung-Hoon Kim wrote:
> >
> >
> >"Hiranya S. Roychowdhury" schrieb:
> >
> >> At 12:32 PM 11/16/99 +1100, Dr Raja Kota wrote:
> >> >Hi Netters,
> >> >
> >> >   Whats the best way to sequence a fragment which is over 1500 bp in
> >> >length and is cloned into a vector.  Any references shall be appreciated.
> >> >
> >> >Thanks in advance,
> >> >
> >> >Raja
> >> >
> >> >
> >>
> >> Send it off to an automated sequencer facility.
> >
> >> If you want to do it yourself, you can try and chop it up into managabloe
> >> pieces that can be sequenced manually.
> >>
> >
> >I think. you don't have to chop it up.
> >Instead, you can use the sequencing primer for your vector. If you're lucky,
> >you can read the hole sequence.
> >Otherwise, make an internal primer and sequence the construct with this primer
> >again.
> >I think, it'll be sufficient for a 1.5 kb fragment.
> >
> >Byung-Hoon Kim
> >Center for Plant Molecular Biology
> >Univ. Tuebingen
> >Germany
> >
>
> I seriously doubt that 1500+ bases can be sequenced *manually* from the
> external primers. I am yet to see a manual sequencing raction that covers
> more than 500 bases, legibly.
> Yes, one can design internal primers from whatever sequence one obtains in
> the first go and continue sequencing from those. However, I consider this
> strategy a terrible waste of money since, in most cases, those nested
> primers are never used again once the sequencing is done.
>
> Dr. Hiranya Sankar Roychowdhury

Sorry, I misunderstood the meaning of 'MANUAL (radioactive) SEQUENCING', because
what we usually do 'manually' is the reaction for automated sequencing. If an
automated fluorescence sequencer is not available, I would send the construct to a
commercial sequencing service. I believe, it's the fastest way to sequence such a
construct.

Byung-Hoon Kim
Center for Plant Molecular Biology
Univ. Tuebingen
Germany






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