sequencing problems

Qunrui Ye qunruiye at
Thu Jun 15 12:23:47 EST 1995


>      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.

Qunrui Ye
Institut fur Veterinarbiochemie               Tel: (41-1)-257-54-79
Universitat Zurich-Irchel                     Fax: (41-1)-362-05-01
Winterthurerstrasse 190
CH-8057 Zurich

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