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Sequencing problems. HELP!

Dr. C.J. Chan cchan at crc.ac.uk
Tue Jul 19 06:37:38 EST 1994

Dear Netters,

I am asking foradvice on the CircumVent Thermo Cycle DNA Sequencing

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!

  C-TJ Chan                               Tel. +44 71 242 9789 Ext 2351        
  Dept of Molecular Genetics,             Fax. +44 71 831 0488                 
  Institute of Child Health,              email cchan at hgmp.mrc.ac.uk
  30 Guilford Street,                                                          
  London WC1N 1EH                                                              
  United Kingdom                                                               

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