qunruiye at vetbio.unizh.ch
Thu Jun 15 12:23:47 EST 1995
In article <01HRMAR7Y82A8WWAS2 at HELIX.MGH.HARVARD.EDU>,
BONVENTRE at HELIX.MGH.HARVARD.EDU wrote:
> I have subcloned 5 fragments of genomic DNA into pBluescript and I
> can not get sequence with the T7orT3 primers. The DNA looks fine on a
> gel and the RI Cut releases the insert nicely. I have subcloned a different
> clone using the same pBlue vector and the sequence is very nice. The
> difficult pieces just give a smear on the gel. I have played with the
> starting concentration to no avail. I also have a completely different
> construct that uses different vector primers and also internal primers
> which give the same smear. In both cases the vectors sequenced as positive
> controls give very good sequence. This has been going on for some time now
> and is very, very frustrating, any help would be appreciated. I have handed
> a couple of the pieces over for sequencing but the results were the same.
> I use Wizard maxi-preps and XL1- Blue cells. Remember there is a construct
> that looks very good that was subjected to all the same steps.
> Thanks in advance
> Eileen O'Leary
I would suggest you try to prepare DNA by QIAgen.
Institut fur Veterinarbiochemie Tel: (41-1)-257-54-79
Universitat Zurich-Irchel Fax: (41-1)-362-05-01
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