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Sequencing problems. HELP!

Ron Kagan rkagan at ewald.mbi.ucla.edu
Thu Jul 21 14:26:01 EST 1994


In article <30gdu2$p9t at mserv1.dl.ac.uk> Dr. C.J. Chan, cchan at crc.ac.uk
writes:
>Dear Netters,
>
>I am asking foradvice on the CircumVent Thermo Cycle DNA Sequencing
>Kit.
>
>Following the recommended protocol and cycling conditions, the control 
>DNA and primer included in the kit worked beautifully. However, my own 
>template is giving 'dirty' results. Only the bottom part of the gel 
>could be read with any confidence. Longer products are weak and have 
>stops in all 4 lanes.
>
>I am using 1.7ug of template and 25ng of primer per reaction. Despite 
>what is said in the manual, I have found that I get no products when I 
>was using less template.
>
>My template was prepared using the Qiagen kit and checking the plasmid
on 
>an agarose gel showed a single nice clean intense band. I do not think I 
>can get better prep that this.
>
>My primers were T3(20 mer) and T7(23 mer). The same tube of primers have 
>worked very well with Sequenase on other templates.
>
>It sounds like a template problem to me. But I am at a loss now as how
to 
>further improve the quality of my template.
>
>I have used Sequenase on this template before but had not joy for similar
>reasons. I get faint bands and stops in all 4 lanes. This is why I
switched
>to cycle sequencing. 
>
>What have I done wrong? Please Help. I need to sequence this thing ASAP!

I would suggest using less primer.  I do cycle sequencing with the USB
delta Taq kit and they reccommend using no more than 0.5 pmoles of primer
per reaction.  They say that excess primer will dilute the signal.  If
you are using 25 ng of primer, that is over 3 pmoles for a 20mer (25,000
pg / 6600 pg/pmole = 3.8 pmole).

You also should try using less tempate, say, 0.05 pmole of plasmid (165
ng for a 5 Kb plasmid).  This works great for 35 cycles of sequencing
with 32-P end-labelled primer.


Ron Kagan


**********************************************************************
"My Kindness is not random, and nothing senseless was ever beautiful."

                                                 - J. Zabriskie
**********************************************************************
Ron Kagan
rkagan at ewald.mbi.ucla.edu



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